Immunoconjugates comprising signal regulatory protein alpha

ABSTRACT

Provided is an immunoconjugate useful in inhibiting tumor growth, and a composition and/or protein mixture comprising the immunoconjugate. Also provided are methods for the production of the immunoconjugate, as well as pharmaceutical uses of the immunoconjugate in inhibiting tumor growth, including but not limited to treatment of cancers.

BACKGROUND

Signal regulatory protein α (SIRPα), also known as SHPS-1, BIT, or CD172a, is an immunoglobulin superfamily protein that binds to the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region for potential negative signaling. SIRPα is particularly abundant in the myeloid-lineage hematopoietic cells such as macrophages and dendritic cell (Reinhold et al., 1997. Costimulation of T cell activation by integrin-associated protein (CD47) is an adhesion-dependent, CD28-independent signaling pathway. J Exp Med 185:1-11). CD47-SIRPα interaction functions as a “don't eat me” signal to ensure that autologous cells are not inappropriately phagocytosed.

However, systematic expression of SIRPα may result in undesired side effects. Further, when expressed alone, SIRPα may have a relatively short in vivo half-life and may not be able to reach the site of action efficiently. Hence, there is a need for novel and effective regulators of the CD47-SIRPα signaling.

SUMMARY

The present disclosure provides an immunoconjugate useful in inhibiting tumor growth, and compositions and/or protein mixture comprising the immunoconjugate. The present disclosure also provides methods for the production of the immunoconjugate, as well as pharmaceutical uses of the immunoconjugate in inhibiting tumor growth, including but not limited to treatment of cancers.

In one aspect, the present disclosure provides an immunoconjugate comprising: a) a targeting moiety, wherein the targeting moiety exhibits binding specificity to a tumor associated antigen; b) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein the first Fc subunit associates with the second Fc subunit to form a dimer; and c) a single copy of a signal-regulatory protein alpha (SIRPα).

In some embodiments, the SIRPα is not a CV1 variant.

In some embodiments, the SIRPα is a human wildtype SIRPα.

In some embodiments, said SIRPα comprises an amino acid sequence as set forth in SEQ ID NO: 122 or 123.

In some embodiments, the targeting moiety comprises an antigen-binding domain of an antibody and said antibody is selected from the group consisting of: anti-EGFRVIII, anti-HER2/neu, anti-PD-L1 and anti-Muc1.

In some embodiments, the antibody is an anti-PD-L1 antibody. For example, the anti-PD-L1 antibody may be selected from the group consisting of Atezolizumab, Avelumab, Durvalumab KN035 and hu56.

In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the IgG is a human IgG1.

In some embodiments, the immunoconjugate is a proteinaceous heterodimer comprising a first member and a second member different from said first member, wherein said first member comprises at least one targeting moiety fused to one of the two subunits of the Fc domain, and said second member comprises said single copy of SIRPα fused to the other one of the two subunits of the Fc domain, and said first Fc subunit associates with said second Fc subunit to form said heterodimer.

In some embodiments, the targeting moiety is directly or indirectly fused to the amino-terminal amino acid of one of the two subunits of the Fc domain, and the single copy of SIRPα is directly or indirectly fused to the amino-terminal amino acid of the other one of the two subunits of the Fc domain.

In some embodiments, the immunoconjugate comprises two of the targeting moiety, which are a first targeting moiety and a second targeting moiety.

In some embodiments, the first targeting moiety is directly or indirectly fused to one of said two subunits of the Fc domain, and said second targeting moiety is directly or indirectly fused to the other one of said two subunits of the Fc domain.

In some embodiments, the immunoconjugate is a proteinaceous heterodimer. The proteinaceous heterodimer may comprise a first member and a second member different from the first member, wherein: the first member may comprise the first targeting moiety fused to one of the two subunits of the Fc domain; the second member may comprise the single copy of SIRPα, the second targeting moiety and the other one of the two subunits of the Fc domain, with the single copy of SIRPα fused to one terminal of the Fc subunit and the second targeting moiety fused to the other terminal of the Fc subunit; and the first Fc subunit associates with the second Fc subunit to form the heterodimer. For example, the first targeting moiety may be directly or indirectly fused to the amino-terminal amino acid of one of the two subunits of the Fc domain, the second targeting moiety may be directly or indirectly fused to the amino-terminal amino acid of the other one of the two subunits of the Fc domain, and the single copy of SIRPα may be directly or indirectly fused to the carboxy-terminal amino acid of the first or the second Fc subunit.

In some embodiments, the first Fc subunit is different from the second Fc subunit, and the Fc domain comprises a modification promoting heterodimerization between the first Fc subunit and the second Fc subunit. For example, the first Fc subunit may comprise a first modification, and the second Fc subunit comprises a second modification, and the first modification comprises an amino acid substitution at 2-5 positions. For example, the first modification may comprise an amino acid substitution at a group of positions selected from any of the following groups: 1) the first modification: Y349 and T366; and the second modification: D356, T366, L368, Y407 and F405; 2) the first modification: Y349, T366 and F405; and the second modification: D356, T366, L368 and Y407; 3) the first modification: Y349, T366 and K409; and the second modification: D356, T366, L368, Y407 and F405; 4) the first modification: Y349, T366, F405, K360 and Q347; and the second modification: D356, T366, L368, Y407 and Q347; 5) the first modification: Y349, T366, F405 and Q347; and the second modification: D356, T366, L368, Y407, K360 and Q347; 6) the first modification: Y349, T366, K409, K360 and Q347; and the second modification: D356, T366, L368, Y407, F405 and Q347; 7) the first modification: Y349, T366, K409 and Q347; and the second modification: D356, T366, L368, Y407, F405, K360 and Q347; 8) the first modification: T366, K409 and K392; and the second modification: T366, L368, Y407, D399 and F405; 9) the first modification: T366 and K409; and the second modification: T366, L368, Y407 and F405; 10) the first modification: T366, K409 and Y349; and the second modification: T366, L368, Y407, F405 and E357; 11) the first modification: T366, K409, Y349 and S354; and the second modification: T366, L368, Y407, F405 and E357; 12) the first modification: T366 and F405; and the second modification: T366, L368, Y407 and K409; 13) the first modification: T366, F405 and D399; and the second modification: T366, L368, Y407, K409 and K392; 14) the first modification: T366, F405 and Y349; and the second modification: T366, L368, Y407, K409 and E357; 15) the first modification: T366, F405, Y349 and S354; and the second modification: T366, L368, Y407, K409 and E357; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

In some embodiments, the single copy of SIRPα is directly or indirectly fused to the second Fc subunit comprising the second modification.

In one aspect, the present disclosure provides an isolated nucleic acid or isolated nucleic acids encoding the immunoconjugate of the present disclosure.

In one aspect, the present disclosure provides a vector or vectors comprising the isolated nucleic acid or isolated nucleic acids of the present disclosure.

In one aspect, the present disclosure provides an isolated host cell comprising the isolated nucleic acid or isolated nucleic acids of the present disclosure, or the vector or vectors of the present disclosure.

In one aspect, the present disclosure provides a method for producing the immunoconjugate or the protein mixture of the present disclosure. The method may comprise (i) culturing the host cell of the present disclosure under conditions to effect expression and formation of the immunoconjugate of the present disclosure, and (ii) harvesting the immunoconjugate formed or the protein mixture comprising the immunoconjugate.

In one aspect, the present disclosure provides a pharmaceutical composition comprising an effective amount of the immunoconjugate of the present disclosure, and optionally a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition further comprises an effective amount of an additional therapeutically active component for cancer treatment. For example, the additional therapeutically active component for cancer treatment may be an agent for chemotherapy. In some embodiments, the agent for chemotherapy is a cytotoxic agent. For example, the cytotoxic agent may comprise doxorubicin.

In one aspect, the present disclosure provides a use of the immunoconjugate, the protein mixture, or the pharmaceutical composition of the present disclosure, in the preparation of a medicament for treating a disease in a subject in need thereof.

In one aspect, the present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the immunoconjugate, the protein mixture, or the pharmaceutical composition of the present disclosure.

In some embodiments, the disease is cancer. For example, the cancer may be selected from the group consisting of: breast cancer, melanoma, and colon cancer.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the application are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present application will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the application are employed, and the accompanying drawings (also “figure” and “FIG.” herein), of which:

FIG. 1a-1b illustrate examples of the immunoconjugate of the present disclosure.

FIG. 2 illustrates the purification result of the immunoconjugate of the present disclosure, as shown by SDS-PAGE analysis.

FIG. 3 illustrates the binding affinity of the immunoconjugate of the present disclosure, as shown by FACS analysis.

FIG. 4 illustrates the results of in vitro pro-phagocytosis activity of the immunoconjugate of the present disclosure.

FIG. 5 illustrates the results of tumor binding affinity of the immunoconjugate of the present disclosure.

FIGS. 6a-6h illustrate the binding affinity of the immunoconjugate of the present disclosure, as shown by flow cytometry results.

FIGS. 7a-7b illustrate the platelets binding result of the immunoconjugate of the present disclosure.

FIGS. 8a-8i illustrate the in vivo anti-tumor efficacy and safety of the immunoconjugate of the present disclosure.

FIG. 9 illustrate the effect of the immunoconjugate of the present disclosure when used in combination with a chemotherapy.

FIG. 10 illustrates the in vivo effect of the immunoconjugate of the present disclosure.

DETAILED DESCRIPTION

While various embodiments of the application have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the application. It should be understood that various alternatives to the embodiments of the application described herein may be employed.

The term “immunoconjugate”, as used herein, generally refers to a proteinaceous molecule formed by the conjugation of one or more antibodies or a fragment thereof to one or more second molecules. The second molecule may be the same or different, and may include for example, effector proteins.

The term “targeting moiety”, as used herein, generally refers to a molecule, complex or aggregate, that binds specifically, selectively or preferentially to a target molecule, cell, particle, tissue or aggregate. For example, a targeting moiety may be an antibody, antigen-binding antibody fragment, bispecific antibody or other antibody-based molecule or compound. Other examples of targeting moieties may include, but are not limited to, aptamers, avimers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins, etc. The terms “targeting moiety” and “binding moiety” are used interchangeably herein.

The term “binding specificity”, as used herein, generally refers to an ability of one substance to bind another substance specifically, and not easily to bind any other substance at random. For example, one protein may bind to another protein specifically due to their specific structures. For example, a targeting moiety may exhibit binding specificity to a corresponding tumor associated antigen.

The term “tumor associated antigen”, as used herein, generally refers to an antigenic substance related to tumor cells, which may have an ability to trigger an immune response in a host or to indicate that the host may have a tumor. For example, a tumor associated antigen may be a protein, a polypeptide, a peptide, or a fragment thereof, which constitutes part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes. A tumor associated antigen peptide may be a peptide that is generated as a result of degradation of the tumor associated antigen in a tumor cell and can induce or activate tumor-specific cytotoxic T lymphocytes upon being expressed on cell surface. In some embodiments, the term “tumor associated antigen” may also refer to biomolecules (e.g., proteins, carbohydrates, glycoproteins, etc.) that are exclusively or preferentially or differentially expressed on a cancer cell and/or are found in association with a cancer cell and thereby provide targets preferential or specific to the cancer. For example, the preferential expression can be preferential expression as compared to any other cell in the organism, or preferential expression within a particular area of the organism (e.g. within a particular organ or tissue).

The term “Fc domain”, as used herein, generally refers to an Fc part or Fc fragment of an antibody heavy chain. For example, it may refer to the carboxyl terminal portion of an immunoglobulin heavy chain constant region, or an analog or portion thereof capable of binding an Fc receptor. As is known, each immunoglobulin heavy chain constant region comprises four or five domains. The domains are named sequentially as follows: CH1-hinge-CH2-CH3(-CH4). CH4 is present in IgM, which has no hinge region. The immunoglobulin heavy chain constant region useful in the present disclosure may comprise an immunoglobulin hinge region, and may also include a CH3 domain. For example, the immunoglobulin heavy chain constant region may comprise an immunoglobulin hinge region, a CH2 domain and a CH3 domain. In some embodiments, the Fc region according to the present disclosure consists of the hinge-CH2-CH3 domain.

The term “Fc subunit”, as used herein, generally refers to a component of an Fc domain. For example, an Fc domain may be formed by two or more members, and each member may be considered as one Fc subunit.

The term “SIRPα”, as used herein, generally refers to a signal regulatory protein α or fragments thereof. SIRPα is a regulatory membrane glycoprotein from SIRP family expressed mainly by myeloid cells and also by stem cells or neurons. A SIRPα may be a human SIRPα, or functional variants thereof.

The term “CV1 variant”, as used herein, generally refers to a consensus variant 1 of SIRPα. It was produced due to nine grafted conservative substitutions onto the predominant wild-type human SIRPα allele, and the CV1 variant can bind human CD 47 with an affinity of 11.1 pM (Kipp Weiskopf et al., Science. 2013 Jul. 5; 341(6141)). Hence, CV1 can competes with endogenous SIRPα for CD47 binding. However, as an Fc-free antagonist, it cannot inhibit cancer growth in vivo. It is found that the synergy between CV1 and other therapeutic mAbs is so dramatic that possibility for overestimation of CD47-SIRPα interaction remains in the presence of ADCC (Yuting Huang et al., J Thorac Dis. 2017 February; 9(2): E168-E174).

The term “single copy”, as used herein, generally refers to the fact that a specific protein or element or feature is present as only one copy in a molecule, a conjugate or a complex.

The term “peptide linker”, as used herein, generally refers to a synthetic amino acid sequence that connects or links two polypeptide sequences, e.g., that link two polypeptide domains. A linker may connect two amino acid sequences via peptide bonds. In some embodiments, a linker of the present disclosure connects a biologically active moiety to a second moiety in a linear sequence. The term “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The peptide also encompasses an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. The peptide may apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The peptide may also include variants on the traditional peptide linkage joining the amino acids making up the polypeptide. Peptides may also include essentially any poly-amino acid including, but not limited to peptide mimetics such as amino acids joined by an ether as opposed to an amide bond.

The term “amino-terminal”, as used herein, generally refers to the free α-amino group on an amino acid at the amino terminal of a peptide or to the α-amino group (amino group when participating in a peptide bond) of an amino acid at any other location within the peptide.

The term “immunoglobulin hinge region”, as used herein, generally refers to the hinge region of an immunoglobulin, for example, an immunoglobulin hinge region may be an IgG1 hinge, an IgG2 hinge, an IgG3 hinge or an IgG4 hinge. The term “IgG1 hinge” is used herein to refer to a hinge region from a native IgG1, such as a native human, mouse, rabbit, etc. IgG1, derivatives of one of the naturally occurring IgG1 hinges, such as derivatives that have at least 95%, 90%, 85%, 80%, 75%, 70%, or 65% amino acid identity with a native IgG1 hinge and retain essential functional characteristics of the IgG1 hinge. The term “hinge region” generally refers to the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region may comprise approximately 25 residues and may be flexible, thus allowing the N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al. J. Immunol. 1998 161:4083).

The term “immunoglobulin” as used herein, generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the κ, λ, α, γ (IgG1, IgG2, IgG3, IgG4), δ, c ε and μ constant region genes, as well as the myriad immunoglobulin variable region genes. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions. In addition to antibodies, immunoglobulins may exist in a variety of other forms including, for example, Fv, Fab, Fab′ and (Fab′)2.

The term “antigen binding domain of an antibody”, as used herein, generally refers to a part of an antibody that participates in antigen binding. An antigen binding domain may be formed by amino acid residues of the N-terminal variable (“V”) regions of a heavy (“H”) chain and/or a light (“L”) chain. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions”. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen binding “surface”. This surface may mediate recognition and binding of the target antigen. The three hypervariable regions of each of the heavy and light chains are referred to as “complementarity determining regions” or “CDRs” and are characterized, for example by Kabat et al. Sequences of proteins of immunological interest, 4^(th) ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, Md. (1987).

The term “Fab”, as used herein, generally refers to a portion (such as an antigen-binding fragment) of an immunoglobulin molecule. An Fab fragment may comprise one light chain and part of a heavy chain with a single antigen-binding site. A Fab fragment may be obtained by papain digestion of an immunoglobulin molecule. For example, a Fab fragment may be composed of one constant and one variable domain of each of the heavy and the light chain. The variable domain may contain the paratope (the antigen-binding site), comprising a set of complementarity determining regions, at the amino terminal end of the immunoglobulin molecule. The enzyme papain may be used to cleave an immunoglobulin molecule into two Fab fragments and one Fc fragment. The enzyme pepsin cleaves below the hinge region, so a F(ab′)2 fragment and a Fc fragment is formed.

The term “domain antibody”, as used herein, generally refers to a single domain antibody, also known as sdAb or Nanobody. A domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than common antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (˜50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (˜25 kDa, two variable domains, one from a light and one from a heavy chain).

The term “ScFv”, as used herein, generally refers to a single-chain antibody fragment. An ScFv may refer to a recombinant single chain polypeptide molecule in which light and heavy chain variable regions of an antibody are connected by a peptide linker. Single chain antibodies (ScFv) generally do not include portions of the Fc region of antibodies that are involved in effector functions and are thus naked antibodies, although methods are known for adding such regions to known ScFv molecules if desired. See Helfrich et al., A rapid and versatile method for harnessing ScFv antibody fragments with various biological functions. J Immunol Methods 237: 131-145 (2000) and de Haard et al., Creating and engineering human antibodies for immunotherapy. Advanced Drug Delivery Reviews 31:5-31 (1998).

The term “anti-EGFR antibody”, as used herein, generally refers to an antibody that specifically or preferentially binds an EGFR. In some cases, and anti-EGFR antibody may bind to a mutated form of EGFR (e.g., EGFR variant III (also known as EGFRvIII), which is the most common extracellular domain mutation of EGFR, this mutation leads to a deletion of exons 2-7 of the EGFR gene, which is characterized by a truncated extracellular domain with ligand-independent constitutive activity). For example, an anti-EGFR antibody may be Cetuximab, Mab806, or antigen binding fragments thereof.

The term “anti-HER2 antibody”, as used herein, generally refers to an antibody that specifically or preferentially binds a HER2/neu receptor. For example, an anti-HER2/neu antibody or anti-HER2 antibody may be Trastuzumab, Pertuzumab, or antigen binding fragments thereof.

The term “anti-PD-L1 antibody”, as used herein, generally refers to an antibody that specifically or preferentially binds to programmed death-ligand 1 (PD-L1). For example, an anti-PD-L1 antibody may be Atezolizumab, Avelumab, Durvalumab, KN035 or hu56. Atezolizumab is a humanized, engineered monoclonal antibody of IgG1 isotype against PD-L1 (from Genentech/Roche). KN035 is a PD-L1 single domain antibody Fc (IgG1 isotype) fusion protein, also named as hu56V2-Fc (as described in CN106397592A), and hu56 is a human single domain antibody against PD-L1 (as described in CN106397592A). And the position of the antibody CDRs is determined according to the antibody Kabat definition. Avelumab is a fully human monoclonal antibody developed by Merck KGaA and Pfizer as a pharmaceutical drug for use in immunotherapy, originally for the treatment of non-small-cell lung carcinoma, and targets the protein PD-L1. Durvalumab is a human immunoglobulin G1 kappa (IgG1K) monoclonal antibody that blocks the interaction of PD-L1 and PD1 (from Medimmune/AstraZeneca). The term “IgG”, as used herein, generally refers to a subtype of an antibody. Each IgG has two antigen binding sites. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in the circulation. Some of the known immunoglobulin genes include the κ, λ, α, and γ (IgG1, IgG2, IgG3, and IgG4).

The term “in frame”, as used herein, generally refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of each original ORF.

The term “proteinaceous”, as used herein, generally refers to a material or molecule that is of, relating to, resembling, or being a polypeptide or a protein. For example, a proteinaceous heterodimer of the present disclosure may be a heterodimer protein, or a heterodimer comprising two or more polypeptides.

The term “heterodimer”, as used herein, generally refers to a molecule (e.g. a proteinaceous molecule) composed of two different members (i.e., two different monomers). The two members of a heterodimer may differ in structure, function, activity and/or composition. For example, the two different members may comprise polypeptides differing in the order, number, or kind of amino acid residues forming these polypeptides. Each of the two different members of a heterodimer may independently comprise one, two or more units, polypeptide chains, or moieties. The two members may aggregate, complex or associate with each other via covalent and/or non-covalent interactions. For example, the two different monomers may associate with each other via interactions between interface amino acid residues from each of the two monomers

The term “modification promoting heterodimerization”, as used herein, generally refers to a modification which promotes the process of heterodimerization. And the term heterodimerization as used herein, generally refers to the process of forming a heterodimer between two different members (e.g., two different polypeptides), such as through complexation, association, or aggregation, with or without formation of covalent bonds between the two different members.

The term “anti-CD47 monoclonal antibody”, as used herein, generally refers to a monoclonal antibody that specifically or preferentially binds to CD47. For example, an anti-CD47 monoclonal antibody may be AB6.12 IgG1. In another example, an anti-CD47 monoclonal antibody may be B6H12.2.

The term “protein mixture”, as used herein, generally refers to a mixture of two or more types of proteins.

The term “homodimer”, as used herein, generally refers to a molecule formed by two identical monomers (e.g., two identical members or subunits). The two monomers may aggregate, complex or associate with each other via covalent and/or non-covalent interactions. For example, the two identical monomers may associate with each other via interactions between interface amino acid residues from each of the two monomers.

The term “about”, as used herein, generally refers to an approximation to a given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. For example, it may refer to a value that is no more than 10% above or below the value being modified by the term. For example, the term “about 5 μg/kg” means a range of 4.5 μg/kg to 5.5 μg/kg. As another example, “about 1 hour” means a range of 48 minutes to 72 minutes.

The term “substantially comprise no”, as used herein, generally refers to that a composition (e.g., a mixture) comprises little or almost none of a specific substance. For example, the specific substance may be present with a percentage of e.g., less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01%.

The term “conditions to effect expression”, as used herein, generally refers to conditions influencing the expression of the immunoconjugate of the present application. In some embodiments, the conditions to effect expression include but not limited to incubation time, temperature, and culture medium, and may depend on cell type and may be readily determined by one of ordinary skill in the art. In some embodiments, during the process of producing the immunoconjugate or the protein mixture of the present disclosure, the host cells are grown in cultures, and in any apparatus that may be used to grow cultures, including fermenters. Cells may be grown as monolayers or attached to a surface. Alternatively, the host cells may be grown in suspension. The cells can be grown in a culture medium that is serum-free. The media can be a commercially available media, such as, but not limited to, Opti-CHO (Invitrogen, Catalogue #12681) supplemented with glutamine, such as 8 mM L-glutamine; RPMI 1640 medium, supplemented with 10% bovine calf serum, 10.5 ng/ml mIL-3 and L-glutamine; or 5% FCS medium.

The term “pharmaceutically acceptable excipient”, as used herein, generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration.

The term “therapeutically active component for cancer treatment”, as used herein, generally refers to any component that has therapeutic activity for the treatment of cancer. For example, a therapeutically active component for cancer treatment may be an agent for chemotherapy.

The term “agent for chemotherapy”, as used herein, generally refers to agents used in a chemotherapy for cancer treatment. For example, the agent for chemotherapy may include but not limited to a cytotoxic agent, an alkylating agent, an antimetabolite agent, an anti-tumor antibiotic agent, and an anti-tumor hormone.

The term “cytotoxic agent”, as used herein, generally refers to an agent that has cytotoxicity. For example, a cytotoxic agent may comprise doxorubicin.

The term “effective amount”, as used herein, generally refers to a dose sufficient to provide concentrations high enough to impart a beneficial effect on the recipient thereof. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated, the severity of the disorder, the activity of the specific component, the route of administration, the rate of clearance, the duration of treatment, the age, body weight, sex, diet, and general health of the subject, and other related factors.

The term “complexed with” as used herein, generally refers to the association (e.g., binding) of one member/subunit with another member/subunit of a molecule (e.g., an antibody). For example, a light chain may be complexed with a heavy chain to form a targeting moiety. In another example, a first Fc subunit may be complexed with a second Fc subunit to form an Fc dimer (an Fc region).

The term “binding specificity” as used herein, generally refers to the ability to specifically bind (e.g., immunoreact with) a given target (while not binding or substantially not binding a non-target). A targeting moiety of the present disclosure may be monospecific and contain one or more binding sites which specifically bind a target or may be multispecific (e.g., bispecific or trispecific) and contain two or more binding sites which specifically bind the same or different targets.

The term “modification” as used herein, generally refers to any manipulation of the peptide backbone (e.g. amino acid sequence) or any post-translational modifications (e.g. glycosylation) of a polypeptide. For example, a modification is in comparison to the sequence of a corresponding wildtype polypeptide. A modification may be a substitution, an addition, and/or a deletion of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more).

The term “fusion protein” as used herein, generally refers to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of a polypeptide fused directly or indirectly (e.g., via a linker) to an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to the former polypeptide or the domain thereof).

The term “C-terminus” as used herein, generally refers to the carboxy terminus of a polypeptide.

The term “N-terminus” as used herein, generally refers to the amino terminus of a polypeptide.

The term “EGFR” as used herein, generally refers to epidermal growth factor receptor. for example, see in Carpenter et al. Ann. Rev. Biochem. 56:881-914 (1987), including naturally occurring mutant forms thereof.

The term “EGFR mutant” as used herein, generally refers to a mutated form of EGFR (e.g., EGFR variant III (also known as EGFRvIII), which is the most common extracellular domain mutation of EGFR, this mutation leads to a deletion of exons 2-7 of the EGFR gene which is characterized by a truncated extracellular domain with ligand-independent constitutive activity.

The term “HER2/neu” as used herein, generally refers to a human HER2 protein, for example, in Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234 (1986) (GenBank accession number X03363).

The term “Muc1” as used herein, generally refers to a glycoprotein encoded by the muc1 gene. Muc1 is mainly present in the epithelial tissues and organs of mammary gland, pancreas, ovary, etc. It is highly expressed on the surface of cancer epithelial cells, and accordingly becomes the target of immune response.

The term “amino acid substitution” as used herein, generally refers to that one amino acid at a specific position of a polypeptide is replaced by another amino acid.

The term “EU index of the KABAT number” as used herein, generally refers to the index of the EU number corresponding to the amino acid sequence according to Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242.

The term “isolated polynucleotide” as used herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from its native environment, or that is artificially synthesized.

Immunoconjugate, Protein Mixtures, Isolated Nucleotides, Vectors and Host Cells

In one aspect, the present disclosure provides an immunoconjugate comprising: a) a targeting moiety, wherein the targeting moiety exhibits binding specificity to a tumor associated antigen; b) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein the first Fc subunit associates with the second Fc subunit to form a dimer; and c) a single copy of a signal-regulatory protein alpha (SIRPα).

The single copy of SIRPα may be directly or indirectly fused to an amino-terminal or carboxy-terminal amino acid of the first Fc subunit or the second Fc subunit of the Fc domain. For example, the single copy of SIRPα may be indirectly fused to the first Fc subunit or the second Fc subunit of the Fc domain through a peptide linker. The linker may be a peptide comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. For example, the linker may comprise 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids), 1-15 amino acids (e.g., 1-11, 12, 13, 14, 15 amino acids), 1-20 amino acids, 1-30 amino acids or more. In some embodiments, the linker comprises an amino acid sequence as set forth in SEQ ID NO: 53, 58, 65, 68 or 71.

For example, the single copy of SIRPα may be fused to the first Fc subunit or the second Fc subunit of the Fc domain in frame.

The SIRPα may be a wild-type signal-regulatory protein alpha or an amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of wild-type signal-regulatory protein alpha or a native or naturally occurring allelic variant of signal-regulatory protein alpha. In some embodiments, the SIRPα is a wild-type mammalian SIRPα, for example, the SIRPα is a wild-type human SIRPα. The amino acid sequence for the mature form of the predominant wild type human SIRPα (SIRPαV2) without a signal peptide may be as set forth in SEQ ID NO: 122. The SIRPα may include a signal peptide. For example, the amino acid sequence of the human SIRPα (SIRPαV2) with a signal peptide may be as set forth in SEQ ID NO: 123.

The targeting moiety may be directly or indirectly fused to an amino-terminal or carboxy-terminal amino acid of the first Fc subunit and/or the second Fc subunit of the Fc domain. For example, the targeting moiety may be indirectly fused to the first Fc subunit and/or the second Fc subunit of the Fc domain through a peptide linker or an immunoglobulin hinge region. The linker may be a peptide comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. For example, the linker may comprise 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids), 1-15 amino acids (e.g., 1-11, 12, 13, 14, 15 amino acids), 1-20 amino acids, 1-30 amino acids or more. For example, the linker may comprise an amino acid sequence as set forth in SEQ ID NO: 53, 58, 65, 68 or 71.

The targeting moiety may comprise an antigen binding domain of an antibody. For example, the antigen binding domain of an antibody may be a Fab moiety, a domain antibody or a ScFv moiety.

The tumor associated antigen may be selected from the group consisting of: EGFRVIII, HER2/neu, PD-L1, and Muc1. For example, the targeting moiety may comprise an antigen-binding domain of an antibody and the antibody may be selected from the group consisting of: anti-EGFRVIII, anti-HER2/neu, anti-PD-L1 and anti-Muc1 antibody.

The antibody may be an anti-EGFRVIII antibody. For example, the anti-EGFRVIII antibody may be Mab 806. The targeting moiety may comprise the heavy chain CDR1-3 of Mab 806, the light chain CDR1-3 of Mab 806, the heavy chain variable region of Mab 806, the light chain variable region of Mab 806, and/or the light chain of Mab 806. For example, the targeting moiety may be a Fab moiety comprising both the heavy chain variable region and the light chain variable region (or the light chain) of Mab 806. In another example, the targeting moiety may be a domain antibody (e.g., a VHH) comprising the heavy chain variable region of Mab 806. In another example, the targeting moiety may be an ScFv moiety comprising the heavy chain variable region and the light chain variable region of Mab 806.

For example, the targeting moiety may comprise heavy chain CDRs having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding heavy chain CDR1-3 of Mab 806. Alternatively or additionally, the targeting moiety may comprise light chain CDRs having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain CDR1-3 of Mab 806. For example, the targeting moiety may comprise a heavy chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding heavy chain variable region of Mab 806. For example, the targeting moiety may comprise a light chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain variable region of Mab 806. For example, the targeting moiety may comprise a light chain having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain of Mab 806.

The heavy chain CDR 1-3 of Mab 806 are as set forth in SEQ ID NO: 90 (CDR1), SEQ ID NO: 91 (CDR2), and SEQ ID NO: 92 (CDR3), respectively. The light chain CDR 1-3 of Mab 806 are as set forth in SEQ ID NO: 86 (CDR1), SEQ ID NO: 87 (CDR2), and SEQ ID NO: 88 (CDR3), respectively. The heavy chain variable region of Mab 806 is as set forth in SEQ ID NO: 93. The light chain variable region of Mab 806 is as set forth in SEQ ID NO: 89.

The antibody may be an anti-HER2 antibody. For example, the anti-HER2 antibody may be Pertuzumab. The targeting moiety may comprise the heavy chain CDR1-3 of Pertuzumab, the light chain CDR1-3 of Pertuzumab, the heavy chain variable region of Pertuzumab, the light chain variable region of Pertuzumab, and/or the light chain of Pertuzumab. For example, the targeting moiety may be a Fab moiety comprising both the heavy chain variable region and the light chain variable region (or the light chain) of Pertuzumab. In another example, the targeting moiety may be a domain antibody (e.g., a VHH) comprising the heavy chain variable region of Pertuzumab. In another example, the targeting moiety may be an ScFv moiety comprising the heavy chain variable region and the light chain variable region of Pertuzumab.

For example, the targeting moiety may comprise heavy chain CDRs having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding heavy chain CDR1-3 of Pertuzumab. For example, alternatively or additionally, the targeting moiety may comprise light chain CDRs having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain CDR1-3 of Pertuzumab. For example, the targeting moiety may comprise a heavy chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding heavy chain variable region of Pertuzumab. For example, the targeting moiety may comprise a light chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain variable region of Pertuzumab. For example, the targeting moiety may comprise a light chain having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain of Pertuzumab.

The heavy chain CDR 1-3 of Pertuzumab are as set forth in SEQ ID NO: 106 (CDR1), SEQ ID NO: 107 (CDR2), and SEQ ID NO: 108 (CDR3), respectively. The light chain CDR 1-3 of Pertuzumab are as set forth in SEQ ID NO: 102 (CDR1), SEQ ID NO: 103 (CDR2), and SEQ ID NO: 104 (CDR3), respectively. The heavy chain variable region of Pertuzumab is as set forth in SEQ ID NO: 109. The light chain variable region of Pertuzumab is as set forth in SEQ ID NO: 105.

In some embodiments, the anti-HER2 antibody is Trastuzumab. The targeting moiety may comprise the heavy chain CDR1-3 of Trastuzumab, the light chain CDR1-3 of Trastuzumab, the heavy chain variable region of Trastuzumab, the light chain variable region of Trastuzumab, and/or the light chain of Trastuzumab. For example, the targeting moiety may be a Fab moiety comprising both the heavy chain variable region and the light chain variable region (or the light chain) of Trastuzumab. In another example, the targeting moiety may be a domain antibody (e.g., a VHH) comprising the heavy chain variable region of Trastuzumab. In another example, the targeting moiety may be an ScFv moiety comprising the heavy chain variable region and the light chain variable region of Trastuzumab.

For example, the targeting moiety may comprise heavy chain CDRs having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding heavy chain CDR1-3 of Trastuzumab. Alternatively or additionally, the targeting moiety may comprise light chain CDRs having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain CDR1-3 of Trastuzumab. For example, the targeting moiety may comprise a heavy chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding heavy chain variable region of Trastuzumab. For example, the targeting moiety may comprise a light chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain variable region of Trastuzumab. For example, the targeting moiety may comprise a light chain having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain of Trastuzumab.

The heavy chain CDR 1-3 of Trastuzumab are as set forth in SEQ ID NO: 98 (CDR1), SEQ ID NO: 99 (CDR2), and SEQ ID NO: 100 (CDR3), respectively. The light chain CDR 1-3 of Trastuzumab are as set forth in SEQ ID NO: 94 (CDR1), SEQ ID NO: 95 (CDR2), and SEQ ID NO: 96 (CDR3), respectively. The heavy chain variable region of Trastuzumab is as set forth in SEQ ID NO: 101. The light chain variable region of Trastuzumab is as set forth in SEQ ID NO: 97.

The antibody may be an anti-Muc1 antibody. For example, the anti-Muc1 antibody may be 5E5 or a humanized version thereof. The targeting moiety may comprise the heavy chain CDR1-3 of 5E5 or a humanized version thereof, the light chain CDR1-3 of 5E5 or a humanized version thereof, the heavy chain variable region of 5E5 or a humanized version thereof, the light chain variable region of 5E5 or a humanized version thereof, and/or the light chain of 5E5 or a humanized version thereof. For example, the targeting moiety may be a Fab moiety comprising both the heavy chain variable region and the light chain variable region (or the light chain) of 5E5 or a humanized version thereof. In another example, the targeting moiety may be a domain antibody (e.g., a VHH) comprising the heavy chain variable region of 5E5 or a humanized version thereof. In another example, the targeting moiety may be an ScFv moiety comprising the heavy chain variable region and the light chain variable region of 5E5 or a humanized version thereof.

For example, the targeting moiety may comprise heavy chain CDRs having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding heavy chain CDR1-3 of 5E5 or a humanized version thereof. Alternatively or additionally, the targeting moiety may comprise light chain CDRs having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain CDR1-3 of 5E5 or a humanized version thereof. For example, the targeting moiety may comprise a heavy chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding heavy chain variable region of 5E5 or a humanized version thereof. For example, the targeting moiety may comprise a light chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain variable region of 5E5 or a humanized version thereof. For example, the targeting moiety may comprise a light chain having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain of 5E5 or a humanized version thereof.

The heavy chain CDR 1-3 of 5E5 are as set forth in SEQ ID NO: 114 (CDR1), SEQ ID NO: 115 (CDR2), and SEQ ID NO: 116 (CDR3), respectively. The light chain CDR 1-3 of 5E5 are as set forth in SEQ ID NO: 110 (CDR1), SEQ ID NO: 111 (CDR2), and SEQ ID NO: 112 (CDR3), respectively. The heavy chain variable region of 5E5 is as set forth in SEQ ID NO: 117. The light chain variable region of 5E5 is as set forth in SEQ ID NO: 113.

The antibody may be an anti-PD-L1 antibody. For example, the anti-PD-L1 antibody may be selected from the group consisting of Atezolizumab, Avelumab, Durvalumab, KN035 and hu56. The targeting moiety may comprise the heavy chain CDR1-3 of Atezolizumab, Avelumab, Durvalumab, KN035 and hu56, the light chain CDR1-3 of Atezolizumab, Avelumab and Durvalumab, the heavy chain variable region of Atezolizumab, Avelumab, Durvalumab, KN035 and hu56, the light chain variable region of Atezolizumab, Avelumab and Durvalumab, and/or the light chain of Atezolizumab, Avelumab and Durvalumab. For example, the targeting moiety may be a Fab moiety comprising both the heavy chain variable region and the light chain variable region (or the light chain) of Atezolizumab, Avelumab and Durvalumab. In another example, the targeting moiety may be a domain antibody (e.g., a V_(H)H) comprising the heavy chain variable region of Atezolizumab, Avelumab, Durvalumab, KN035 and hu56. In another example, the targeting moiety may be an ScFv moiety comprising the heavy chain variable region and the light chain variable region of Atezolizumab, Avelumab and Durvalumab.

For example, the anti-PD-L1 antibody may be selected from the group consisting of Atezolizumab, Avelumab, Durvalumab, KN035 and hu56. For example, the targeting moiety may comprise heavy chain CDR 1-3 having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding heavy chain CDR1-3 of Atezolizumab, Avelumab, Durvalumab, KN035 and hu56. Alternatively or additionally, the targeting moiety may comprise light chain CDR 1-3 having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain CDR1-3 of Atezolizumab, Avelumab and Durvalumab. For example, the targeting moiety may comprise a heavy chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding heavy chain variable region of Atezolizumab, Avelumab, Durvalumab, KN035 and hu56. For example, the targeting moiety may comprise a light chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain variable region of Atezolizumab, Avelumab and Durvalumab. For example, the targeting moiety may comprise a light chain having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that comprised in the corresponding light chain of Atezolizumab, Avelumab and Durvalumab.

The heavy chain CDR 1-3 of hu56 are as set forth in SEQ ID NO: 118 (CDR1), SEQ ID NO: 119 (CDR2), and SEQ ID NO: 120 (CDR3), respectively. The heavy chain variable region of hu56 is as set forth in SEQ ID NO: 121.

The heavy chain CDR 1-3 of KN035 are as set forth in SEQ ID NO: 118 (CDR1), SEQ ID NO: 119 (CDR2), and SEQ ID NO: 120 (CDR3), respectively. The heavy chain variable region of hu56 is as set forth in SEQ ID NO: 121.

The heavy chain CDR 1-3 of Atezolizumab are as set forth in SEQ ID NO: 128 (CDR1), SEQ ID NO: 129 (CDR2), and SEQ ID NO: 130 (CDR3), respectively. The light chain CDR 1-3 of Atezolizumab are as set forth in SEQ ID NO: 124 (CDR1), SEQ ID NO: 125 (CDR2), and SEQ ID NO: 126 (CDR3), respectively. The heavy chain variable region of Atezolizumab is as set forth in SEQ ID NO: 131. The light chain variable region of Atezolizumab is as set forth in SEQ ID NO: 127.

The heavy chain CDR 1-3 of Avelumab are as set forth in SEQ ID NO: 136 (CDR1), SEQ ID NO: 137 (CDR2), and SEQ ID NO: 138 (CDR3), respectively. The light chain CDR 1-3 of Avelumab are as set forth in SEQ ID NO: 132 (CDR1), SEQ ID NO: 133 (CDR2), and SEQ ID NO: 134 (CDR3), respectively. The heavy chain variable region of Avelumab is as set forth in SEQ ID NO: 139. The light chain variable region of Avelumab is as set forth in SEQ ID NO: 135.

The heavy chain CDR 1-3 of Durvalumab are as set forth in SEQ ID NO: 144 (CDR1), SEQ ID NO: 145 (CDR2), and SEQ ID NO: 146 (CDR3), respectively. The light chain CDR 1-3 of Durvalumab are as set forth in SEQ ID NO: 140 (CDR1), SEQ ID NO: 141 (CDR2), and SEQ ID NO: 142 (CDR3), respectively. The heavy chain variable region of Durvalumab is as set forth in SEQ ID NO: 147. The light chain variable region of Durvalumab is as set forth in SEQ ID NO: 143.

In the immunoconjugate of the present disclosure, the Fc domain may be an IgG Fc domain. For example, the IgG may be selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. In some embodiments, the IgG is a human IgG1, and the Fc domain is a human IgG1 Fc domain (wildtype or modified).

In the immunoconjugate of the present disclosure, first Fc subunit of the Fc domain may be different from the second Fc subunit, and the Fc domain may comprise a modification promoting heterodimerization between the first Fc subunit and the second Fc subunit. For example, the first Fc subunit may comprise a first modification, and the second Fc subunit comprises a second modification.

The first modification may comprise an amino acid substitution at position T366, and an amino acid substitution at one or more positions selected from the group consisting of: Y349, F405, K409, D399, K360, Q347, K392 and S354, wherein the position of the amino acid is determined according to the EU index of the KABAT number. For example, the amino acid substitution comprised by the first modification may be selected from the group consisting of Y349C, Y349D, D399S, F405K, K360E, K409A, K409E, Q347E, Q347R, S354D, K392D and T366W.

The first modification may comprise an amino acid substitution at 2-7 positions (e.g., 2-6, 2-5, 2-4, 2-3, or 2 positions). For example, the first modification may comprise an amino acid substitution at a group of positions selected from any of the following groups: 1) Y349 and T366; 2) Y349, T366 and F405; 3) Y349, T366 and K409; 4) Y349, T366, F405, K360 and Q347; 5) Y349, T366, F405 and Q347; 6) Y349, T366, K409, K360 and Q347; 7) Y349, T366, K409 and Q347; 8) T366, K409 and K392; 9) T366 and K409; 10) T366, K409, Y349 and S354; 11) T366 and F405; 12) T366, F405 and D399; and 13) T366, F405, Y349 and S354. For example, the first modification may comprise a group of amino acid substitutions selected from any of the following groups: 1) Y349C and T366W; 2) Y349C, T366W and F405K; 3) Y349C, T366W and K409E; 4) Y349C, T366W and K409A; 5) Y349C, T366W, F405K, K360E and Q347E; 6) Y349C, T366W, F405K and Q347R; 7) Y349C, T366W, K409A, K360E and Q347E; 8) Y349C, T366W, K409A and Q347R; 9) T366W, K409A and K392D; 10) T366W and K409A; 11) T366W, K409A and Y349D; 12) T366W, K409A, Y349D and S354D; 13) T366W and F405K; 14) T366W, F405K and D399S; 15) T366W, F405K and Y349D; and 16) T366W, F405K, Y349D and S354D.

The second modification may comprise amino acid substitutions at positions T366, L368 and Y407, as well as an amino acid substitution at one or more positions selected from the group consisting of D356, D399, E357, F405, K360, K392, K409 and Q347, wherein the position of the amino acid is determined according to the EU index of the KABAT number. For example, the amino acid substitution comprised by the second modification may be selected from the group consisting of D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A and Y407V.

The second modification may comprise an amino acid substitution at 4-8 positions (e.g., 4-7, 4-6, 4-5, or 4 positions). For example, the second modification may comprise an amino acid substitution at a group of positions selected from any of the following groups: 1) D356, T366, L368, Y407 and F405; 2) D356, T366, L368 and Y407; 3) D356, T366, L368, Y407 and Q347; 4) D356, T366, L368, Y407, K360 and Q347; 5) D356, T366, L368, Y407, F405 and Q347; 6) D356, T366, L368, Y407, F405, K360 and Q347; 7) T366, L368, Y407, D399 and F405; 8) T366, L368, Y407 and F405; 9) T366, L368, Y407, F405 and E357; 10) T366, L368, Y407 and K409; 11) T366, L368, Y407, K409 and K392; and 12) T366, L368, Y407, K409 and E357. For example, the second modification may comprise a group of amino acid substitutions selected from any of the following groups: 1) D356C, T366S, L368A, Y407V and F405K; 2) D356C, T366S, L368A and Y407V; 3) D356C, T366S, L368A, Y407V and Q347R; 4) D356C, T366S, L368A, Y407V, K360E and Q347E; 5) D356C, T366S, L368A, Y407V, F405K and Q347R; 6) D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 7) T366S, L368A, Y407V, D399S and F405K; 8) T366S, L368G, Y407A and F405K; 9) T366S, L368A, Y407V, F405K and E357A; 10) T366S, L368A, Y407V and K409A; 11) T366S, L368A, Y407V, K409A and K392D; 12) T366S, L368G, Y407A and K409A; 13) T366S, L368A, Y407V, K409A and E357A.

For example, the first Fc subunit may comprise the first modification, the second Fc subunit may comprise the second modification, the first modification and the second modification may comprise an amino acid substitution at a group of positions selected from any of the following groups: 1) the first modification: Y349 and T366; and the second modification: D356, T366, L368, Y407 and F405; 2) the first modification: Y349, T366 and F405; and the second modification: D356, T366, L368 and Y407; 3) the first modification: Y349, T366 and K409; and the second modification: D356, T366, L368, Y407 and F405; 4) the first modification: Y349, T366, F405, K360 and Q347; and the second modification: D356, T366, L368, Y407 and Q347; 5) the first modification: Y349, T366, F405 and Q347; and the second modification: D356, T366, L368, Y407, K360 and Q347; 6) the first modification: Y349, T366, K409, K360 and Q347; and the second modification: D356, T366, L368, Y407, F405 and Q347; 7) the first modification: Y349, T366, K409 and Q347; and the second modification: D356, T366, L368, Y407, F405, K360 and Q347; 8) the first modification: T366, K409 and K392; and the second modification: T366, L368, Y407, D399 and F405; 9) the first modification: T366 and K409; and the second modification: T366, L368, Y407 and F405; 10) the first modification: T366, K409 and Y349; and the second modification: T366, L368, Y407, F405 and E357; 11) the first modification: T366, K409, Y349 and S354; and the second modification: T366, L368, Y407, F405 and E357; 12) the first modification: T366 and F405; and the second modification: T366, L368, Y407 and K409; 13) the first modification: T366, F405 and D399; and the second modification: T366, L368, Y407, K409 and K392; 14) the first modification: T366, F405 and Y349; and the second modification: T366, L368, Y407, K409 and E357; 15) the first modification: T366, F405, Y349 and S354; and the second modification: T366, L368, Y407, K409 and E357; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

For example, the first Fc subunit may comprise the first modification, the second Fc subunit may comprise the second modification, wherein the first modification and the second modification may comprise a group of amino acid substitutions selected from any of the following groups: 1) the first modification: Y349C and T366W; and the second modification: D356C, T366S, L368A, Y407V and F405K; 2) the first modification: Y349C, T366W and F405K; and the second modification: D356C, T366S, L368A and Y407V; 3) the first modification: Y349C, T366W and K409E; and the second modification: D356C, T366S, L368A, Y407V and F405K; 4) the first modification: Y349C, T366W and K409A; and the second modification: D356C, T366S, L368A, Y407V and F405K; 5) the first modification: Y349C, T366W, F405K, K360E and Q347E; and the second modification: D356C, T366S, L368A, Y407V and Q347R; 6) the first modification: Y349C, T366W, F405K and Q347R; and the second modification: D356C, T366S, L368A, Y407V, K360E and Q347E; 7) the first modification: Y349C, T366W, K409A, K360E and Q347E; and the second modification: D356C, T366S, L368A, Y407V, F405K and Q347R; 8) the first modification: Y349C, T366W, K409A and Q347R; and the second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) the first modification: T366W, K409A and K392D; and the second modification: T366S, L368A, Y407V, D399S and F405K; 10) the first modification: T366W and K409A; and the second modification: T366S, L368G, Y407A and F405K; 11) the first modification: T366W, K409A and Y349D; and the second modification: T366S, L368A, Y407V, F405K and E357A; 12) the first modification: T366W, K409A, Y349D and S354D; and the second modification: T366S, L368A, Y407V, F405K and E357A; 13) the first modification: T366W and F405K; and the second modification: T366S, L368A, Y407V and K409A; 14) the first modification: T366W, F405K and D399S; and the second modification: T366S, L368A, Y407V, K409A and K392D; 15) the first modification: T366W and F405K; and the second modification: T366S, L368G, Y407A and K409A; 16) the first modification: T366W, F405K and Y349D; and the second modification: T366S, L368A, Y407V, K409A and E357A; 17) the first modification: T366W, F405K, Y349D and S354D; and the second modification: T366S, L368A, Y407V, K409A and E357A; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

In some embodiments, the first Fc subunit comprises the first modification, the second Fc subunit comprises the second modification, the first modification comprises the amino acid substitutions T366W and K409A, and the second modification comprises the amino acid substitutions T366S, L368G, Y407A and F405K, wherein the position of the amino acid is determined according to the EU index of the KABAT number.

In some embodiments, the single copy of SIRPα is directly or indirectly fused to the second Fc subunit comprising the second modification.

The amino acid sequence of the first Fc subunit may be selected from SEQ ID NO: 39, 43, 47, 51, 54, 56, 59, 76, 80 or 84.

The amino acid sequence of the second Fc subunit may be selected from SEQ ID NO: 66 or 72.

The immunoconjugate may comprise one or more of said targeting moieties.

In some embodiments, the immunoconjugate comprises only one targeting moiety. In some embodiments, the immunoconjugate comprises two or more targeting moieties.

For example, the immunoconjugate may be a proteinaceous heterodimer comprising a first member and a second member different from the first member. The first member may comprise the targeting moiety fused to one of the two subunits of the Fc domain, and the second member may comprise the single copy of SIRPα fused to the other one of the two subunits of the Fc domain. The first Fc subunit may associate with the second Fc subunit to form the heterodimer.

For example, the targeting moiety may be directly or indirectly (e.g., via said linker) fused to the amino-terminal amino acid of one of the two subunits of the Fc domain, and the single copy of SIRPα may be directly or indirectly (e.g., via said linker) fused to the amino-terminal amino acid of the other one of the two subunits of the Fc domain.

In some embodiments, the immunoconjugate comprises two of the targeting moiety, which are a first targeting moiety and a second targeting moiety. The first targeting moiety and the second targeting moiety may exhibit binding specificity to different tumor associated antigens or to the same tumor associated antigen. For example, the first targeting moiety and the second targeting moiety may exhibit binding specificity to the same tumor associated antigen. In some embodiments, the first targeting moiety and the second targeting moiety are the same.

The first targeting moiety and the second targeting moiety may be fused to each other directly or indirectly (e.g., via a linker).

When the immunoconjugate comprises two of the targeting moiety, the first targeting moiety may be directly or indirectly fused to an amino-terminal or carboxy-terminal amino acid of one of the two subunits of the Fc domain, and the second targeting moiety may be directly or indirectly fused to an amino-terminal or carboxy-terminal amino acid of the other one of the two subunits of the Fc domain.

For example, the immunoconjugate may be a proteinaceous heterodimer. The proteinaceous heterodimer may comprise a first member and a second member different from the first member. The first member may comprise the first targeting moiety fused to one of the two subunits of the Fc domain, and the second member may comprise the single copy of SIRPα, the second targeting moiety and the other one of the two subunits of the Fc domain, with the single copy of SIRPα fused to one terminal of the Fc subunit and the second targeting moiety fused to the other terminal of the Fc subunit. The first Fc subunit may associate with the second Fc subunit to form the heterodimer.

For example, the first targeting moiety may be directly or indirectly fused to the amino-terminal amino acid of one of the two subunits of the Fc domain, the second targeting moiety may be directly or indirectly fused to the amino-terminal amino acid of the other one of the two subunits of the Fc domain, and the single copy of SIRPα may be directly or indirectly fused to the carboxy-terminal amino acid of the first or the second Fc subunit. For example, the first targeting moiety may be fused to the amino-terminal amino acid of the first Fc subunit, the second targeting moiety may be fused to the amino-terminal amino acid of the second Fc subunit, and the single copy of SIRPα may be fused to the carboxy-terminal amino acid of the second Fc subunit.

In some embodiments, when the immunoconjugate comprises two of the targeting moiety, both the first targeting moiety and the second targeting moiety may be directly or indirectly fused to the same one of the two subunits of the Fc domain.

For example, the first targeting moiety and the second targeting moiety may be directly or indirectly (e.g., via a linker) fused to each other to form a dimer of the targeting moiety. Then, the dimer of the targeting moiety may be directly or indirectly fused to one of the two subunits of the Fc domain.

The immunoconjugate may be a proteinaceous heterodimer. The proteinaceous heterodimer may comprise a first member and a second member different from the first member. The first member may comprise both the first targeting moiety and the second targeting moiety. The first targeting moiety and the second targeting moiety may be fused to the same one of the two subunits of the Fc domain (for example, the first targeting moiety and the second targeting moiety may be directly or indirectly (e.g., via a linker) fused to each other to form a dimer of the targeting moiety. Then, the dimer of the targeting moiety may be directly or indirectly fused to one of the two subunits of the Fc domain). The second member may comprise the single copy of SIRPα and the other one of the two subunits of the Fc domain. The single copy of SIRPα may be fused to one terminal of the Fc subunit. The first Fc subunit may associate with the second Fc subunit to form the heterodimer.

For example, both the first targeting moiety and the second targeting moiety may be directly or indirectly fused to the amino-terminal amino acid of the same one of the two subunits of the Fc domain (for example, the first targeting moiety and the second targeting moiety may be directly or indirectly (e.g., via a linker) fused to each other to form a dimer of the targeting moiety. Then, the dimer of the targeting moiety may be directly or indirectly fused to the amino-terminal amino acid of one of the two subunits of the Fc domain). The single copy of SIRPα may be directly or indirectly fused to the amino-terminal amino acid of the other one of the two subunits of the Fc domain. For example, the first targeting moiety and the second targeting moiety may be fused to the amino-terminal amino acid of the first Fc subunit (for example, the first targeting moiety and the second targeting moiety may be directly or indirectly (e.g., via a linker) fused to each other to form a dimer of the targeting moiety. Then, the dimer of the targeting moiety may be directly or indirectly fused to the amino-terminal amino acid of the first Fc subunit), and the single copy of SIRPα may be fused to the amino-terminal amino acid of the second Fc subunit.

The first Fc subunit may comprise the first modification as defined in the present disclosure and the second Fc subunit may comprise the second modification as defined in the present disclosure.

The immunoconjugate of the present disclosure may have a % red blood cell (RBC) binding mean fluorescence intensity (MFI) of no more than about 20%, no more than about 19%, no more than about 18%, no more than about 17%, no more than about 16%, no more than about 15%, no more than about 14%, no more than about 13%, no more than about 12%, no more than about 11%, no more than about 10%, no more than about 8%, no more than about 6%, no more than about 4%, no more than about 2% or less when % RBC binding MFI to a reference immunoconjugate is calibrated at 100%, wherein the reference immunoconjugate is a homodimer protein consisting of two identical members, each member of the homodimer protein consists of a single copy of SIRPα fused via a peptide linker to the amino-terminal amino acid of a human IgG1 Fc subunit (also referred to as SIRPα-Fc in the present application).

The immunoconjugate may also have a % platelet binding mean fluorescence intensity (MFI) of no more than about 65%, no more than about 60%, no more than about 59%, no more than about 58%, no more than about 57%, no more than about 56%, no more than about 55%, no more than about 54%, no more than about 53%, no more than about 52%, no more than about 51%, no more than about 50%, no more than about 45%, no more than about 40%, no more than about 30%, no more than about 25% or less when % platelet binding MFI to a reference immunoconjugate is calibrated at 100%, wherein the reference immunoconjugate is a homodimer protein consisting of two identical members, each member of the homodimer protein consists of a single copy of SIRPα fused via a peptide linker to the amino-terminal amino acid of a human IgG1 Fc subunit (also referred to as SIRPα-Fc IgG1 in the present application).

In some embodiments, each member of the reference immunoconjugate SIRPα-Fc IgG1 has an amino acid sequence as set forth in SEQ ID NO. 69.

The immunoconjugate may have a % red blood cell (RBC) binding mean fluorescence intensity (MFI) of no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2.5%, no more than about 2%, no more than about 1.5%, no more than about 1% or less when % RBC binding MFI to a reference molecule is calibrated at 100%, wherein the reference molecule is the anti-CD47 monoclonal antibody AB6.12 IgG1.

The immunoconjugate may have a % platelet binding mean fluorescence intensity (MFI) of no more than about 50%, no more than about 45%, no more than about 46%, no more than about 45%, no more than about 44%, no more than about 43%, no more than about 42%, no more than about 41%, no more than about 40%, no more than about 39%, no more than about 38%, no more than about 37%, no more than about 36%, no more than about 35%, no more than about 34%, no more than about 33%, no more than about 32%, no more than about 31%, no more than about 30%, no more than about 25%, no more than about 20% or less when % platelet binding MFI to a reference molecule is calibrated at 100%, wherein the reference molecule is the anti-CD47 monoclonal antibody AB6.12 IgG1.

In another aspect, the present disclosure provides an isolated nucleic acid or isolated nucleic acids encoding the immunoconjugate of the present disclosure. The isolated nucleic acids may comprise one or more nucleic acid molecules, with each encoding the immunoconjugate of the present disclosure or a fragment thereof. For example, the isolated nucleic acids may comprise at least two nucleic acid molecules, with one encoding the targeting moiety fused to the first Fc subunit and the second one encoding the single copy of SIRPα fused to the second Fc subunit. In some cases, the second one may encode a polypeptide comprising a second targeting moiety fused to the second Fc subunit and the single copy of SIRPα. In some cases, the isolated nucleic acids may comprise at least three nucleic acid molecules, with one encoding a light chain of the targeting moiety, a second one encoding a heavy chain of the targeting moiety (comprising the first Fc subunit), and a third one encoding the single copy of SIRPα fused to the second Fc subunit. In some cases, the isolated nucleic acids may comprise at least four nucleic acid molecules, with one encoding a light chain of a first targeting moiety, a second one encoding a heavy chain of the first targeting moiety (comprising the first Fc subunit), a third one encoding a light chain of a second targeting moiety, and a fourth one encoding a heavy chain of the second targeting moiety (comprising the second Fc subunit) fused to the single copy of SIRPα.

The isolated nucleic acid or isolated nucleic acids may be synthesized using recombinant techniques well known in the art. For example, the isolated nucleic acid or isolated nucleic acids may be synthesized with an automated DNA synthesizer.

Standard recombinant DNA and molecular cloning techniques include those described by Sambrook, J., Fritsch, E. F. and Maniatis, T Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M L. Bennan, and L. W Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987). Briefly, the subject nucleic acids can be prepared from genomic DNA fragments, cDNAs, and RNAs, all of which can be extracted directly from a cell or recombinantly produced by various amplification processes including but not limited to PCR and RT-PCR.

Direct chemical synthesis of nucleic acids typically involves sequential addition of 3′-blocked and 5′-blocked nucleotide monomers to the terminal 5′-hydroxyl group of a growing nucleotide polymer chain, wherein each addition is effected by nucleophilic attack of the terminal 5′-hydroxyl group of the growing chain on the 3′-position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like. See for example, Matteuci et al., Tet. Lett. 521:719 (1980); U.S. Pat. No. 4,500,707 to Caruthers et al.; and U.S. Pat. Nos. 5,436,327 and 5,700,637 to Southern et al.

In another aspect, the present disclosure provides a vector or vectors comprising the isolated nucleic acid or isolated nucleic acids.

The vector may be any linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Non-limiting examples of a viral vector may include a retrovirus, an adenovirus and an adeno-associated virus. In some embodiments, the vector is an expression vector, e.g. a phage display vector.

An expression vector may be suitable for use in particular types of host cells and not others. For example, the expression vector can be introduced into the host organism, which is then monitored for viability and expression of any genes/polynucleotides contained in the vector.

The expression vector may also contain one or more selectable marker genes that, upon expression, confer one or more phenotypic traits useful for selecting or otherwise identifying host cells that carry the expression vector. Non-limiting examples of suitable selectable markers for eukaryotic cells include dihydrofolate reductase and neomycin resistance.

The subject vectors can be introduced into a host cell stably or transiently by a variety of established techniques. For example, one method involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, for example calcium phosphate, may also be used following a similar procedure. In addition, electroporation (that is, the application of current to increase the permeability of cells to nucleic acids) may be used. Other examples of transformation methods include microinjection, DEAE dextran mediated transformation, and heat shock in the presence of lithium acetate. Lipid complexes, liposomes, and dendrimers may also be employed to transfect the host cells.

Upon introduction of the heterologous sequence into a host cell, a variety of methods can be practiced to identify the host cells into which the subject vectors have been introduced. One exemplary selection method involves sub-culturing individual cells to form individual colonies, followed by testing for expression of the desired protein product. Another method entails selecting host cells containing the heterologous sequence based upon phenotypic traits conferred through the expression of selectable marker genes contained within the expression vector.

For example, the introduction of various heterologous sequences of the disclosure into a host cell can be confirmed by methods such as PCR, Southern blot or Northern blot hybridization. For example, nucleic acids can be prepared from the resultant host cells, and the specific sequences of interest can be amplified by PCR using primers specific for the sequences of interest. The amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, followed by staining with ethidium bromide, SYBR Green solution or the like, or detection of DNA with a UV detection. Alternatively, nucleic acid probes specific for the sequences of interest can be employed in a hybridization reaction. The expression of a specific gene sequence can be ascertained by detecting the corresponding mRNA via reverse-transcription coupled with PCR, Northern blot hybridization, or by immunoassays using antibodies reactive with the encoded gene product. Exemplary immunoassays include but are not limited to ELISA, radio-immunoassays, and sandwich immunoassays.

Furthermore, the introduction of various heterologous sequences of the disclosure into a host cell can be confirmed by the enzymatic activity of an enzyme (e.g., an enzymatic marker) that the heterologous sequence encodes. The enzyme can be assayed by a variety of methods known in the art. In general, the enzymatic activity can be ascertained by the formation of the product or conversion of a substrate of an enzymatic reaction that is under investigation. The reaction can take place in vitro or in vivo.

In another aspect, the present disclosure provides an isolated host cell comprising the isolated nucleic acid or isolated nucleic acids of the present disclosure or the vector or vectors of the present disclosure.

The cell may express the immunoconjugate and/or the protein mixture of the present disclosure, the isolated nucleic acid or nucleic acids encoding the immunoconjugate and/or the protein mixture of the present disclosure. The cell may be a eukaryotic cell or a prokaryotic cell. An appropriate cell may be transformed or transfected with the nucleic acid(s) or vector(s) of the present disclosure, and utilized for the expression and/or secretion of the immunoconjugate and/or protein mixtures. For example, the cell may be E. coli cells, other bacterial host cells, yeast cells, or various higher eukaryotic cells.

In another aspect, the present disclosure provides a method for producing the immunoconjugate of the present disclosure, comprising (i) culturing the host cell under conditions to effect expression and formation of the immunoconjugate, and (ii) harvesting the immunoconjugate formed.

In one aspect, the present disclosure provides a protein mixture comprising the immunoconjugate of the present disclosure. The immunoconjugate may be a proteinaceous heterodimer formed by a first member and a second member. The protein mixture may comprise a first homodimer formed by two identical copies of the first member, wherein each of the first members comprises the targeting moiety fused to the first Fc subunit; a second homodimer formed by two identical copies of the second member, wherein each of the second members comprises the single copy of SIRPα fused to the second Fc subunit; wherein the immunoconjugate of the present disclosure is present in the protein mixture with a percentage of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%.

In the protein mixture, the percentage of the second homodimer may be less than the percentage of the first homodimer. For example, the percentage of the second homodimer may be at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1% or at most about 0.5%. For example, the protein mixture may substantially comprise none of the second homodimer.

The protein mixture may be obtained directly from the host cell of the present disclosure. For example, the protein mixture may be obtained from the host cell without further protein purification.

In another aspect, the present disclosure provides a method for producing the protein mixture, comprising (i) culturing the host cell under conditions to effect expression and formation of the immunoconjugate, and (ii) harvesting the protein mixture comprising the immunoconjugate.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a pharmaceutical composition comprising an effective amount of the immunoconjugate, and optionally a pharmaceutically acceptable excipient.

The pharmaceutical composition may further comprise an effective amount of an additional therapeutically active component for cancer treatment. For example, the additional therapeutically active component for cancer treatment may be an agent for chemotherapy. For example, the agent for chemotherapy may be a cytotoxic agent. For example, the cytotoxic agent may be doxorubicin. Each of the active components may be present in the pharmaceutical composition in a pharmaceutically active amount.

Described below are non-limiting exemplary pharmaceutical compositions and methods for preparing the same.

The pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. The pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption.

Pharmaceutical compositions of the disclosure suitable for oral administration can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such dosage forms can be prepared by any of the methods of pharmacy, but all methods typically include the step of bringing the active ingredient into association with the carrier, which constitutes one or more other ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

An immunoconjugate or a protein mixture of the present disclosure can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.

When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.

The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

Surfactant which can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. A mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.

Within the aforementioned group, ionic surfactants include, by way of example: lecithins, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.

The composition may include a solubilizer to ensure good solubilization and/or dissolution of the immunoconjugate or the protein mixture of the present disclosure and to minimize precipitation of the immunoconjugate or protein mixture of the present disclosure. This can be especially important for compositions for non-oral use, e.g., compositions for injection. A solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

Examples of suitable solubilizers include, but are not limited to, the following: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol and mixtures thereof. The amount of solubilizer that can be included is not particularly limited. The amount of a given solubilizer may be limited to a bioacceptable amount, which may be readily determined by one of skill in the art.

The composition can further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

The pharmaceutical compositions of the present disclosure may comprise a therapeutically effective amount of the active agent (e.g., the immunoconjugate or the protein mixture of the present disclosure). A therapeutically effective amount is an amount of the subject pharmaceutical composition capable of preventing and/or curing (at least partially) a condition or disorder (e.g., cancer) and/or any complications thereof in a subject suffering from or having a risk of developing said condition or disorder. The specific amount/concentration of the active agent comprised may vary according to the method of administration and the need of a patient, and can be determined based on e.g., volume, viscosity, and/or body weight of a patient etc. For example, an appropriate dosage may be about 0.1 mg or 1 mg/kg/day to about 50 mg/kg/day; sometimes, the dosage can be even higher. For example, the dosage applied may be from about 3 mg/kg/day to about 3.5 mg/kg/day, from 3.5 mg/kg/day to about 7.2 mg/kg/day, from about 7.2 mg/kg/day to about 11.0 mg/kg/day, from about 11.0 mg/kg/day to about 15.0 mg/kg/day. For example, the dosage applied may be from about 10 mg/kg/day to about 50 mg/kg/day, for example, from about 20 mg to about 50 mg per day, administered twice/day. It shall be understood that these specific doses may be conveniently adjusted by a skilled person in the art (e.g., a doctor or a pharmacist) based on conditions of a specific patient, formulation, and/or disease.

Medical Use and Methods of Treatment

In one aspect, the present disclosure provides the immunoconjugate, the protein mixture, or the pharmaceutical composition of the present disclosure, for treating a disease in a subject in need thereof.

In another aspect, the present disclosure provides a use of the immunoconjugate, the protein mixture, or the pharmaceutical composition of the present disclosure in the preparation of a medicament, wherein the medicament is for treating a disease in a subject in need thereof.

In one aspect, the present disclosure provides a use of the immunoconjugate, or the protein mixture in combination with the additional therapeutically active component (such as an agent for chemotherapy, for example, the cytotoxic agent, e.g., doxorubicin) in the preparation of a medicament for treating a disease in a subject in need thereof.

In another aspect, the present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the immunoconjugate, the protein mixture, or the pharmaceutical composition of the present disclosure.

In a further aspect, the present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the immunoconjugate, or the protein mixture of the present disclosure, and further administering to the subject an effective amount of the additional therapeutically active component (such as an agent for chemotherapy, for example, the cytotoxic agent, e.g., doxorubicin). The immunoconjugate, or the protein mixture of the present disclosure and the additional therapeutically active component may be administered concurrently or sequentially.

The disease may be cancer. For example, the cancer may comprise tumor cells with elevated expression of CD47. For example, the cancer may be selected from the group consisting of: lung cancer, bladder cancer, gastric cancer, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, glioma, glioblastoma, esophageal cancer, leiomyosarcoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic syndrome, breast cancer, melanoma, and colon cancer. In some embodiments, the cancer is selected from the group consisting of: breast cancer, melanoma, and colon cancer.

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EXAMPLES

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application, and are not intended to limit the scope of what the applicants regard as their application nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., s or sec, second(s); min, minute(s); h or hr, hour(s); i.p., intraperitoneal(ly) and the like.

Example 1 Protein Preparation 1.1 Fc Modifications

Amino acid modifications (e.g., amino acid substitutions) were made to the interface residues of human IgG1 Fc domain to obtain the following groups of modifications (as shown in table 1 below), chain A is also referred to as Fc9 or the first Fc subunit, and chain B is also referred to as Fc6 or the second Fc subunit in the present disclosure:

TABLE 1 Groups of amino acid modifications Group Fc Chain Modifications SEQ ID NO 1 A Y349C + T366W 1 B D356C + T366S + L368A + Y407V + F405K 2 2 A Y349C + T366W + F405K 3 B D356C + T366S + L368A + Y407V 4 3 A Y349C + T366W + K409E 5 B D356C + T366S + L368A + Y407V + F405K 2 4 A Y349C + T366W + K409A 6 B D356C + T366S + L368A + Y407V + F405K 2 5 A Y349C + T366W + F405K + K360E + Q347E 7 B D356C + T366S + L368A + Y407V + Q347R 8 6 A Y349C + T366W + F405K + Q347R 9 B D356C + T366S + L368A + Y407V + K360E + Q347E 10 7 A Y349C + T366W + K409A + K360E + Q347E 11 B D356C + T366S + L368A + Y407V + F405K + Q347R 12 8 A Y349C + T366W + K409A + Q347R 13 B D356C + T366S + L368A + Y407V + F405K + K360E + Q347E 14 9 A T366W + K409A + K392D 15 B T366S + L368A + Y407V + D399S + F405K 16 10 A T366W + K409A 17 B T366S + L368G + Y407A + F405K 18 11 A T366W + K409A + Y349D 19 B T366S + L368A + Y407V + F405K + E357A 20 12 A T366W + K409A + Y349D + S354D 21 B T366S + L368A + Y407V + F405K + E357A 20 13 A T366W + F405K 22 B T366S + L368A + Y407V + K409A 23 14 A T366W + F405K + D399S 24 B T366S + L368A + Y407V + K409A + K392D 25 15 A T366W + F405K 22 B T366S + L368G + Y407A + K409A 26 16 A T366W + F405K + Y349D 27 B T366S + L368A + Y407V + K409A + E357A 28 17 A T366W + F405K + Y349D + S354D 29 B T366S + L368A + Y407V + K409A + E357A 28

Subsequently, formation of heterodimer proteins comprising the groups of modifications listed in Table 1 above were examined using a ScFv-Fc/Fc system, as explained in detail below.

First of all, human immunoglobulin gamma1 (IgG1) constant region amino acid sequence was obtained from the database Uniprot (P01857), to get wildtype human IgG1-Fc region amino acid sequence (SEQ ID NO: 30). The polynucleotide fragment encoding wild type human IgG1-Fc was obtained by RT-PCR from human PBMC total RNA (SEQ ID NO: 31, named as the Fc gene fragment). A polynucleotide fragment encoding a mouse kappaIII signal peptide (SEQ ID NO: 32) was added to the 5′ end of the Fc gene by overlapping PCR, and then subcloned into the vector pcDNA4 (Invitrogen, Cat V86220), to obtain a recombinant expression vector for expressing human IgG1-Fc in mammalian cells.

A nucleic acid molecule encoding a ScFv-Fc fusion protein (SEQ ID NO: 33) was synthesized, wherein the ScFv refers to an anti-HER2 single chain antibody, the amino acid sequence of the ScFv-Fc fusion protein is as set forth in SEQ ID NO: 34. The ScFv-Fc gene fragment was then subcloned into the vector pcDNA4 (Invitrogen, Cat V86220), to obtain a recombinant expression vector for expressing the ScFv-Fc fusion protein in mammalian cells.

In some cases, a polypeptide of a variable region of a camel single domain antibody (VhH) was fused to the N terminal of the Fc gene fragment to obtain a fusion gene fragment (as set forth in SEQ ID NO: 35) encoding the fusion protein VhH-Fc (as set forth in SEQ ID NO: 36). It was then subcloned into the vector pcDNA4 (Invitrogen, Cat V86220), to obtain a recombinant expression vector for expressing the fusion protein VhH-Fc in mammalian cells.

Then, the amino acid modifications as listed in Table 1 above were respectively introduced into the ScFv-Fc (groups 1-17), the VhH-Fc (groups 2, 4, 9-12, 14, 15 and 17), and the Fc gene fragment (groups 1-8) by overlapping PCR, wherein chain A refers to the Fc subunit in ScFv-Fc and chain B refers to the independent Fc subunit or the Fc subunit in VhH-Fc. The gene fragments with amino acid modifications were respectively subcloned into the vector pcDNA4 (Invitrogen, Cat V86220), to obtain recombinant expression vectors for expressing the modified ScFv-Fc fusion proteins, the modified Fc proteins, and the modified VhH-Fc fusion proteins in mammalian cells.

Then, suspend-cultured HEK293 cells (ATCC CRL-1573™) were transfected with the constructed expression vectors with PEI. For each group, the expression vector expressing the A chain (ScFv-Fc fusion protein) and that expressing the B chain (Fc protein or VhH-Fc fusion protein) were co-transfected at a ratio of 1:1. After culturing for 5-6 days, supernatant of the transient expression products was collected, and the expression products comprising corresponding protein heterodimers were preliminarily purified using ProteinA affinity chromatography. Each of the preliminarily purified expression products comprises the homodimer protein ScFv-Fc/ScFv-Fc, the homodimer protein Fc/Fc (or the homodimer protein VhH-Fc/VhH-Fc) and the heterodimer protein ScFv-Fc/Fc (or the heterodimer protein ScFv-Fc/VhH-Fc), present in various percentages, respectively. Since the molecular weight of these proteins (i.e., the homodimers and the heterodimers) are different, their corresponding percentage could be determined according to corresponding band intensities reflected on non-reduced SDS-PAGE gels. The intensities were quantified and the results are summarized in tables 2-5 below.

TABLE 2 Percentage of homodimer proteins and heterodimer proteins in expression products ScFv-Fc ScFv-Fc/Fc Fc Group homodimer (%) heterodimer (%) homodimer (%) 1 24 58 18 2 10 70 20 3 25 57 18 4 10 77 13

TABLE 3 Percentage of homodimer proteins and heterodimer proteins in expression products ScFv-Fc ScFv-Fc/Fc Fc Group homodimer (%) heterodimer (%) homodimer (%) 2 17 60 23 5 14 72 14 6 14 62 24 4 21 69 10 7 24 64 12 8 21 71 8

TABLE 4 Percentage of homodimer proteins and heterodimer proteins in expression products ScFv-Fc ScFv-Fc/VhH-Fc VhH-Fc Group homodimer (%) heterodimer (%) homodimer (%) 4 13 68 19 9 7 80 13 10 15 85 0 11 14 83 3 12 10 84 6

TABLE 5 Percentage of homodimer proteins and heterodimer proteins in expression products ScFv-Fc ScFv-Fc/VhH-Fc VhH-Fc Group homodimer (%) heterodimer (%) homodimer (%) 2 9 64 27 14 6 81 13 15 5 88 7 17 9 84 7

As can be seen from tables 2-5 above, all groups of modifications promoted heterodimer formation very effectively. For illustrative purposes, the modifications in group 10 (modifications in chain A: T366W+K409A; modifications in chain B: T366S+L368G+Y407A+F405K) were used in the following examples to generate the immunoconjugate or the protein mixtures of the present disclosure.

1.2 Preparation of T-Fc9, T-LC and T-HC

Full length amino acid sequences of the heavy chain and light chain of Trastuzumab were obtained according to U.S. Pat. No. 7,879,325B2 (incorporated herein by reference). Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Nucleic acid molecules encoding the light chain of Trastuzumab (T-LC) were then synthesized. The amino acid sequence of T-LC is as set forth in SEQ ID NO: 37, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 38. Nucleic acid molecules encoding the heavy chain of Trastuzumab (T-HC) were then synthesized. The amino acid sequence of T-HC is as set forth in SEQ ID NO: 156, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 157. Then, point mutations (T366W and K409A) were introduced into the polynucleotide sequences encoding the Fc region of Trastuzumab heavy chain gene, and nucleic acid molecules encoding the modified Trastuzumab heavy chain were synthesized (referred to herein as T-Fc9), the corresponding polypeptide encoded was named as T-Fc9. The amino acid sequences of T-Fc9 is as set forth in SEQ ID NO: 39, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO:40.

1.3 Preparation of P-Fc9 and P-LC

Full length amino acid sequences of the heavy chain and light chain of Pertuzumab were obtained according to U.S. Pat. No. 7,879,325B2 (incorporated herein by reference). Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Nucleic acid molecules encoding the light chain of Pertuzumab (P-LC) were then synthesized. The amino acid sequence of P-LC is as set forth in SEQ ID NO:41, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 42. Then, point mutations (T366W and K409A) were introduced into the polynucleotide sequences encoding the Fc region of Pertuzumab heavy chain gene, and nucleic acid molecules encoding the modified Pertuzumab heavy chain were synthesized (referred to herein as P-Fc9), the corresponding polypeptide encoded was named as P-Fc9. The amino acid sequences of P-Fc9 is as set forth in SEQ ID NO: 43, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 44.

1.4 Preparation of Mab806-Fc9 and Mab806-LC

Full length amino acid sequences of the heavy chain and light chain of Mab806 were obtained according to U.S. Pat. No. 7,589,180B2 (incorporated herein by reference). Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Nucleic acid molecules encoding the light chain of Mab806 (Mab806-LC) were then synthesized. The amino acid sequence of Mab806-LC is as set forth in SEQ ID NO:45, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 46. Then, point mutations (T366W and K409A) were introduced into the polynucleotide sequences encoding the Fc region of Mab806 heavy chain gene, and nucleic acid molecules encoding the modified Mab806 heavy chain were synthesized (referred to herein as Mab806-Fc9), the corresponding polypeptide encoded was named as Mab806-Fc9. The amino acid sequences of Mab806-Fc9 is as set forth in SEQ ID NO: 47, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 48.

1.5 Preparation of 5E5-Fc9 and 5E5-LC

Full length amino acid sequences of the heavy chain and light chain of 5E5 were obtained according to U.S. Pat. No. 8,440,798B2 (incorporated herein by reference). Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Nucleic acid molecules encoding the light chain of 5E5 (5E5-LC) were then synthesized. The amino acid sequence of 5E5-LC is as set forth in SEQ ID NO: 49, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 50. Then, point mutations (T366W and K409A) were introduced into the polynucleotide sequences encoding the Fc region of 5E5 heavy chain gene, and nucleic acid molecules encoding the modified 5E5 heavy chain were synthesized (referred to herein as 5E5-Fc9), the corresponding polypeptide encoded was named as 5E5-Fc9. The amino acid sequences of 5E5-Fc9 is as set forth in SEQ ID NO: 51, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 52.

1.6 Preparation of hu56-Fc9

Full length amino acid sequences of the heavy chain variable region of hu56 were obtained according to CN106397592A. Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Then, amino acid sequences of human IgG1-Fc (i.e., residue 104 to residue 330 of P01857) were obtained according to the amino acid sequences of human immunoglobulin γ1 (IgG1) constant region (P01857) from the protein database Uniprot. Afterwards, point mutations (T366W and K409A) were introduced into the IgG1-Fc fragment, and the polypeptide obtained thereby is referred to as Fc9. Then, a linker sequence “GAP” (SEQ ID NO: 53) was added to the N-terminus of the Fc9, to obtain linker-Fc9. The corresponding DNA sequence encoding it was then designed using online tool DNAworks (helixweb.nih.gov/dnaworks/). Polynucleotide sequences encoding the heavy chain variable region of hu56 were added to the 5′ end of the polynucleotide sequences encoding the linker-Fc9, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein hu56-Fc9. The amino acid sequence of hu56-Fc9 is as set forth in SEQ ID NO: 54, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 55.

1.7 Preparation of hu56di-Fc9

Full length amino acid sequences of the heavy chain variable region of hu56 were obtained according to CN106397592A. Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Two copies of the heavy chain variable region of hu56 were linked by a linker “GAP” (SEQ ID NO: 53) to obtain hu56di. Then, amino acid sequences of human IgG1-Fc (i.e., residue 104 to residue 330 of P01857) were obtained according to the amino acid sequences of human immunoglobulin γ1 (IgG1) constant region (P01857) from the protein database Uniprot. Afterwards, point mutations (T366W and K409A) were introduced into the IgG1-Fc fragment, and the polypeptide obtained thereby is referred to as Fc9. Then, a linker sequence “EPKSS” (SEQ ID NO: 58) was added to the N-terminus of the Fc9, to obtain linker-Fc9. The corresponding DNA sequence encoding it was then designed using online tool DNAworks (helixweb.nih.gov/dnaworks/). Polynucleotide sequences encoding hu56di were added to the 5′ end of the polynucleotide sequences encoding the linker-Fc9, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein hu56di-Fc9. The amino acid sequence of hu56di-Fc9 is as set forth in SEQ ID NO:56, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 57.

1.8 Preparation of mPD-L1-Fc9

An anti-mouse PD-L1 ScFv molecule (B50-6-ScFv) was obtained according to CN105777906A, and a nucleic acid molecule encoding a mPD-L1-ScFv-Fc fusion protein was synthesized. Nucleic acid molecules encoding the heavy chain of mPD-L1-ScFv-Fc were then synthesized. The amino acid sequence of mPD-L1-ScFv-Fc is as set forth in SEQ ID NO: 158, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 159. Then, point mutations (T366W and K409A) were introduced into the nucleic acid molecule encoding the mPD-L1-ScFv-Fc fusion protein to obtain mPD-L1-ScFv-Fc9, the corresponding polypeptide encoded was named as mPD-L1-Fc9, and the amino acid sequence thereof is as set forth in SEQ ID NO:59, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 60.

1.9 Preparation of CD47-muFc

The amino acid sequence of the human CD47 extracellular domain (i.e., the 19th residue to the 141th residue in Q08722) was obtained according to the amino acid sequence of human CD47 (Q08722) in the protein database Uniprot. According to the amino acid sequence of the constant region of murine immunoglobulin gamma1 (P01868) in the protein database Uniprot, the amino acid sequence of muFc (i.e., the 112th residue to the 213th residue in P01868) was obtained. The DNAworks Online Tool (http://helixweb.nih.gov) was used to design the corresponding coding DNA sequence to obtain the CD47-muFc fusion protein gene, and the DNA fragment was obtained by artificial synthesis. The synthetic gene sequences were cloned into commercial vector pcDNA4/myc-HisA (Invitrogen, V863-20) by Fermentas HindIII and EcoRI. The recombinant plasmids were then sequenced for confirmation, thereby obtaining the plasmid DNA of pcDNA4-CD47-muFc. The amino acid sequences of CD47-muFc is as set forth in SEQ ID NO: 61, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 62.

1.10 Preparation of CD47-EGFP

The full length amino acid sequence of the human CD47 was obtained according to the amino acid sequence of human CD47 (Q08722) in the protein database Uniprot. According to the amino acid sequence of the Enhanced Green Fluorescent Protein, EGFP (C5MKY7) in the protein database Uniprot, the amino acid sequence of EGFP was obtained. The DNAworks Online Tool (http://helixweb.nih.gov) was used to design the corresponding coding DNA sequence to obtain the CD47-EGFP fusion protein gene, and the DNA fragment was obtained by artificial synthesis. The synthetic gene sequences were cloned into commercial vector pcDNA4/myc-HisA (Invitrogen, V863-20) by Fermentas HindIII and EcoRI. The recombinant plasmids were sequenced for confirmation, thereby obtaining the plasmid DNA of pcDNA4-CD47-EGFP. The amino acid sequences of CD47-EGFP is as set forth in SEQ ID NO: 63, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 64.

1.11 Preparation of SIRPα-Fc6

First of all, sequence information of human SIRPα (NM 080792.2) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequences encoding it were obtained. Then, amino acid sequences of human IgG1-Fc (i.e., residue 104 to residue 330 of P01857) were obtained according to the amino acid sequences of human immunoglobulin γ1 (IgG1) constant region (P01857) from the protein database Uniprot. Afterwards, point mutations (T366S, L368G, Y407A and F405K) were introduced into the IgG1-Fc fragment, and the polypeptide obtained thereby is referred to as Fc6. Then, a linker sequence “GGGGS” (SEQ ID NO: 65) was added to the N-terminus of the Fc6, to obtain linker-Fc6. The corresponding DNA sequence encoding it was then designed using online tool DNAworks (helixweb.nih.gov/dnaworks/). Polynucleotide sequences encoding human SIRPα were added to the 5′ end of the polynucleotide sequences encoding the linker-Fc6, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein human SIRPα-Fc6. The amino acid sequence of human SIRPα-Fc6 (SIRPα-Fc6) is as set forth in SEQ ID NO:66, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 67.

1.12 Preparation of SIRPα-Fc IgG1

First of all, sequence information of human SIRPα (NM_080792.2) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequences encoding it were obtained. Then, amino acid sequences of human IgG1-Fc (i.e., residue 104 to residue 330 of P01857) were obtained according to the amino acid sequences of human immunoglobulin γ1 (IgG1) constant region (P01857) from the protein database Uniprot. Then, a linker sequence “GAPGG” (SEQ ID NO: 68) was added to the N-terminus of the IgG1-Fc, to obtain linker-IgG1-Fc. The corresponding DNA sequence encoding it was then designed using online tool DNAworks (helixweb.nih.gov/dnaworks/). Polynucleotide sequences encoding human SIRPα were added to the 5′ end of the polynucleotide sequences encoding the linker-IgG1-Fc, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein human SIRPα-Fc IgG1. The amino acid sequence of human SIRPα-Fc IgG1 (short for SIRPα-Fc IgG1) is as set forth in SEQ ID NO: 69, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 70.

1.13 Preparation of hu56-Fc6-SIRPα

Full length amino acid sequences of the heavy chain variable region of hu56 were obtained according to CN106397592A. Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Then, amino acid sequences of human IgG1-Fc (i.e., residue 104 to residue 330 of P01857) were obtained according to the amino acid sequences of human immunoglobulin γ1 (IgG1) constant region (P01857) from the protein database Uniprot. Afterwards, point mutations (T366S, L368G, Y407A and F405K) were introduced into the IgG1-Fc fragment, and the polypeptide obtained thereby is referred to as Fc6. Then, a linker sequence “GGGGS” (SEQ ID NO: 65) was added to the N-terminus of the Fc6, to obtain linker-Fc6, and a linker sequence “(GGGGS)₃” (SEQ ID NO: 71) was added to the C-terminus of the linker-Fc6, to obtain linker-Fc6-linker. The sequence information of human SIRPα (NM_080792.2) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequences encoding it were obtained. Polynucleotide sequences encoding human SIRPα were added to the 3′ end of the polynucleotide sequences encoding the linker-Fc6-linker, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein linker-Fc6-SIRPα. Then, the heavy chain variable region of hu56 was fused to the N-terminus of linker-Fc6-SIRPα, to obtain hu56-Fc6-SIRPα. The amino acid sequence of hu56-Fc6-SIRPα is as set forth in SEQ ID NO: 72, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 73.

1.14 Preparation of ate-Fc9 and ate-LC

Full length amino acid sequences of the heavy chain and light chain of atezolizumab were obtained from http://www.imgt.org/mAb-DB/index. Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Nucleic acid molecules encoding the light chain of atezolizumab (ate-LC) were then synthesized. The amino acid sequence of ate-LC is as set forth in SEQ ID NO: 74, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO:75. Then, point mutations (T366W and K409A) were introduced into the polynucleotide sequences encoding the Fc region of amatuximab heavy chain gene, and nucleic acid molecules encoding the modified atezolizumab heavy chain were synthesized (referred to herein as ate-Fc9), the corresponding polypeptide encoded was named as ate-Fc9. The amino acid sequences of ate-Fc9 is as set forth in SEQ ID NO: 76, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 77.

1.15 Preparation of ave-Fc9 and ave-LC

Full length amino acid sequences of the heavy chain and light chain of avelumab were obtained from http://www.imgt.org/mAb-DB/index. Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Nucleic acid molecules encoding the light chain of avelumab (ave-LC) were then synthesized. The amino acid sequence of ave-LC is as set forth in SEQ ID NO: 78, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 79. Then, point mutations (T366W and K409A) were introduced into the polynucleotide sequences encoding the Fc region of avelumab heavy chain gene, and nucleic acid molecules encoding the modified avelumab heavy chain were synthesized (referred to herein as ave-Fc9), the corresponding polypeptide encoded was named as ave-Fc9. The amino acid sequences of ave-Fc9 is as set forth in SEQ ID NO: 80, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 81.

1.16 Preparation of dur-Fc9 and dur-LC

Full length amino acid sequences of the heavy chain and light chain of durvalumab were obtained from http://www.imgt.org/mAb-DB/index. Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Nucleic acid molecules encoding the light chain of durvalumab (dur-LC) were then synthesized. The amino acid sequence of dur-LC is as set forth in SEQ ID NO: 82, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 83. Then, point mutations (T366W and K409A) were introduced into the polynucleotide sequences encoding the Fc region of durvalumab heavy chain gene, and nucleic acid molecules encoding the modified durvalumab heavy chain were synthesized (referred to herein as dur-Fc9), the corresponding polypeptide encoded was named as dur-Fc9. The amino acid sequences of dur-Fc9 is as set forth in SEQ ID NO: 84, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 85.

1.17 Preparation of CV1-Fc6 and CV1-Fc

First of all, sequence information and the synthesis approach of CV1 were described in Weiskopf K. et. al. Engineered SIRPα variants as immunotherapeutic adjuvants to anti-cancer antibodies, Science, 2013, 341(6141): 88-91. Then, amino acid sequences of human IgG1-Fc (i.e., residue 104 to residue 330 of P01857) were obtained according to the amino acid sequences of human immunoglobulin γ1 (IgG1) constant region (P01857) from the protein database Uniprot. Afterwards, point mutations (T366S, L368G, Y407A and F405K) were introduced into the IgG1-Fc fragment, and the polypeptide obtained thereby is referred to as Fc6. Then, a linker sequence “GGGGS” (SEQ ID NO: 65) was added to the N-terminus of the Fc6, to obtain linker-Fc6. The corresponding DNA sequence encoding it was then designed using online tool DNAworks (helixweb.nih.gov/dnaworks/). Polynucleotide sequences encoding CV1 were added to the 5′ end of the polynucleotide sequences encoding the linker-Fc6, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein human CV1-Fc6. The amino acid sequence of CV1-Fc6 is as set forth in SEQ ID NO:148, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 149.

Amino acid sequences of human IgG1-Fc (i.e., residue 104 to residue 330 of P01857) were obtained according to the amino acid sequences of human immunoglobulin γ1 (IgG1) constant region (P01857) from the protein database Uniprot. Then, a linker sequence “GAPGG” (SEQ ID NO: 68) was added to the N-terminus of the IgG1-Fc, to obtain linker-IgG1-Fc. The corresponding DNA sequence encoding it was then designed using online tool DNAworks (helixweb.nih.gov/dnaworks/). Polynucleotide sequences encoding human CV1 were added to the 5′ end of the polynucleotide sequences encoding the linker-IgG1-Fc, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein human CV1-Fc. The amino acid sequence of human CV1-Fc is as set forth in SEQ ID NO: 154, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 155.

1.18 Preparation of mCD47-ScFv-Fc6

A anti-mouse CD47 ScFv molecule (miap-301-ScFv) was was synthesized. Nucleic acid molecules encoding the heavy chain of mCD47-ScFv-Fc were then synthesized. The amino acid sequence of mCD47-ScFv-Fc is as set forth in SEQ ID NO: 160, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 161. Then, point mutations (T366S, L368G, Y407A and F405K) were introduced into the nucleic acid molecule encoding the mCD47-ScFv-Fc fusion protein, to obtain mCD47-ScFv-Fc6, the corresponding polypeptide encoded was named as mCD47-ScFv-Fc6, and the amino acid sequence thereof is as set forth in SEQ ID NO:162, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 163.

1.19 Preparation of mSIRPα-Fc6 and mSIRPα-Fc

First of all, sequence information of mouse SIRPα (NM_001177647.2) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequences encoding it were obtained. Then, amino acid sequences of human IgG1-Fc (i.e., residue 104 to residue 330 of P01857) were obtained according to the amino acid sequences of human immunoglobulin γ1 (IgG1) constant region (P01857) from the protein database Uniprot. Then, a linker sequence “GAPGG” (SEQ ID NO: 68) was added to the N-terminus of the IgG1-Fc, to obtain linker-IgG1-Fc. The corresponding DNA sequence encoding it was then designed using online tool DNAworks (helixweb.nih.gov/dnaworks/). Polynucleotide sequences encoding mSIRPα were added to the 5′ end of the polynucleotide sequences encoding the linker-IgG1-Fc, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein mSIRPα-Fc. The amino acid sequence of mSIRPα-Fc is as set forth in SEQ ID NO: 152, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 153. Afterwards, point mutations (T366S, L368G, Y407A and F405K) were introduced into the IgG1-Fc fragment, and the polypeptide obtained thereby is referred to as Fc6. Then, a linker sequence “GGGGS” (SEQ ID NO: 65) was added to the N-terminus of the Fc6, to obtain linker-Fc6. The corresponding DNA sequence encoding it was then designed using online tool DNAworks (helixweb.nih.gov/dnaworks/). Polynucleotide sequences encoding mouse SIRPα were added to the 5′ end of the polynucleotide sequences encoding the linker-Fc6, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein mouse SIRPα-Fc6. The amino acid sequence of mouse SIRPα-Fc6 (mSIRPα-Fc6) is as set forth in SEQ ID NO:150, and the polynucleotide sequence encoding it is as set forth in SEQ ID NO: 151.

1.20 Preparation of AB6.12-HC and AB6.12-LC

Full length amino acid sequences of the heavy chain and light chain of AB6.12 were obtained according to WO2014123580A (incorporated herein by reference). Then, corresponding DNA sequences encoding these amino acid sequences were obtained using online tool DNAworks (helixweb.nih.gov/dnaworks/). Nucleic acid molecules encoding the light chain of AB6.12 (AB6.12-LC) were then synthesized. The amino acid sequence of AB6.12-LC is as set forth in SEQ ID NO: 166, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 167. Nucleic acid molecules encoding the heavy chain of AB6.12 (AB6.12-HC) were then synthesized. The amino acid sequence of AB6.12-HC is as set forth in SEQ ID NO: 164, and the corresponding polynucleotide sequence encoding it is as set forth in SEQ ID NO: 165.

Example 2 Construction of Recombinant Plasmids

The nucleic acid molecules (encoding T-Fc9, P-Fc9, Mab806-Fc9, 5E5-Fc9, hu56-Fc9, hu56di-Fc9, ate-Fc9, ave-Fc9, dur-Fc9, mPD-L1-Fc9, T-LC (Trastuzumab light chain), P-LC (Pertuzumab light chain), Mab806-LC (Mab806 light chain), 5E5-LC (5E5 light chain), AB6.12-LC (AB6.12 light chain), ate-LC, ave-LC, dur-LC, SIRPα-Fc6, SIRPα-Fc IgG1, CV1-Fc6, hu56-Fc6-SIRPα, mSIRPα-Fc6, T-HC (Trastuzumab heavy chain), AB6.12-HC (AB6.12 heavy chain), CV1-Fc, scFv-Fc and mCD47-scFv-Fc6, respectively) obtained according to Example 1 were digested with HindIII and EcoRI (Takara), and then sub-cloned into the vector pcDNA4/myc-HisA (Invitrogen, V863-20), respectively. The plasmids obtained were verified by sequencing, and the correct recombinant plasmids were named as: pcDNA4-T-Fc9, pcDNA4-P-Fc9, pcDNA4-Mab806-Fc9, pcDNA4-5E5-Fc9, pcDNA4-hu56-Fc9, pcDNA4-hu56di-Fc9, pcDNA4-ate-Fc9, pcDNA4-ave-Fc9, pcDNA4-dur-Fc9, pcDNA4-mPD-L1-Fc9, pcDNA4-T-LC, pcDNA4-P-LC, pcDNA4-Mab806-LC, pcDNA4-5E5-LC, pcDNA4-AB6.12-LC, pcDNA4-ate-LC, pcDNA4-ave-LC, pcDNA4-dur-LC, pcDNA4-SIRPα-Fc6, pcDNA4-SIRPα-Fc IgG1, pcDNA4-CV1-Fc6, pcDNA4-hu56-Fc6-SIRPα, pcDNA4-mSIRPα-Fc6, pcDNA4-T-HC, pcDNA4-AB6.12-HC, pcDNA4-CV1-Fc, pcDNA4-scFv-Fc and pcDNA4-mCD47-scFv-Fc6, respectively.

Example 3 Expression and Purification of Proteinaceous Heterodimers

Two days before transfection, 12×600 mL suspension domesticated HEK293 (ATCC, CRL-1573™) cells were prepared for transient transfection, the cells were seeded at a density of 0.8×10⁶ cells/ml. Two days later, three aliquots of cell suspension were centrifuged, and then resuspended in 600 mL Freestyle293 culture medium.

The recombinant expression vectors obtained from Example 2 were divided into the following groups:

Group 1: pcDNA4-T-Fc9 (200 μg)+pcDNA4-T-LC (200m)+pcDNA4-SIRPα-Fc6(200m)

Group 2: pcDNA4-P-Fc9 (200 μg)+pcDNA4-P-LC (200m)+pcDNA4-SIRPα-Fc6(200m)

Group 3: pcDNA4-Mab806-Fc9 (200 μg)+pcDNA4-Mab806-LC (200m)+pcDNA4-SIRPα-Fc6(200 μg)

Group 4: pcDNA4-5E5-Fc9(200m)+pcDNA4-5E5-LC (200m)+pcDNA4-SIRPα-Fc6(200 μg)

Group 5: pcDNA4-hu56di-Fc9(200m)+pcDNA4-SIRPα-Fc6(200 μg)

Group 6: pcDNA4-mPD-L1-Fc9(200m)+pcDNA4-mSIRPα-Fc6(200 μg)

Group 7: pcDNA4-SIRPα-Fc IgG1 (200 μg) Group 8: pcDNA4-CD47-muFc (200m)

Group 9: pcDNA4-CD47-EGFP (200m)

Group 10: pcDNA4-hu56-Fc9(200m)+pcDNA4-hu56-Fc6-SIRPα(200m)

Group 11: pcDNA4-ate-Fc9(200 μg)+pcDNA4-ate-LC (200 μg)+pcDNA4-SIRPα-Fc6(200m)

Group 12: pcDNA4-ave-Fc9(200 μg)+pcDNA4-ave-LC (200 μg)+pcDNA4-SIRPα-Fc6(200 μg)

Group 13: pcDNA4-dur-Fc9(200 μg)+pcDNA4-dur-LC (200 μg)+pcDNA4-SIRPα-Fc6(200 μg)

Group 14: pcDNA4-hu56di-Fc9(200 μg)+pcDNA4-CV1-Fc6(200m)

Group 15: pcDNA4-mPD-L1-Fc9(200 μg)+pcDNA4-CV1-Fc6(200m)

Group 16: pcDNA4-T-Fc9 (200 μg)+pcDNA4-T-LC (200 μg)+pcDNA4-mSIRPα-Fc6(200m)

Group 17: pcDNA4-mPD-L1-scFv-Fc9 (200 μg)+pcDNA4-mCD47-scFv-Fc6 (200m)

Group 18: pcDNA4-mSIRPα-Fc (200m)

Group 19: pcDNA4-T-HC (200 μg)+pcDNA4-T-LC (200m)

Group 20: pcDNA4-AB6.12-HC (200 μg)+pcDNA4-AB6.12-LC (200m)

Group 21: pcDNA4-mPD-L1-scFv-Fc (200m)

Group 22: pcDNA4-mCD47-scFv-Fc (200 μg)

Group 23: pcDNA4-CV1-Fc (200 μg)

All proteins were made in transiently transfected 293F cells. Briefly, FreeStyle 293F cells (Invitrogen) were grown in 293F medium (Invitrogen), transfected with non-linearized plasmid DNA and 293Fectin reagent (Invitrogen) and grown in shaker flask batches in volumes 80-100 mL/flask at 37° C., 5% CO₂ for 6 days. All proteins were purified by one-step protein A chromatography. The quality of each protein was determined by SDS-PAGE. Similarly, the expression and purification results of the other immunoconjugates of the present application were verified and confirmed with SDS-PAGE.

The immunoconjugates thus obtained are named as (from Group 1 to Group 23, respectively): Tmab-SIRPα, Pmab-SIRPα, Mab806-SIRPα, 5E5-SIRPα, hu56di-SIRPα, mPD-L1-mSIRPα, SIRPα-Fc IgG1, CD47-muFc, CD47-EGFP, hu56-Fc6-SIRPα, ate-SIRPα, ave-SIRPα, dur-SIRPα, hu56di-CV1, mPD-L1-CV1, Tmab-mSIRPα, mPD-L1-mCD47, mSIRPα-Fc, KN034(Herceptin), AB6.12 IgG1, a-mPD-L1, a-mCD47 and CV1-Fc.

In FIG. 2, lane 1 was loaded with SIRPα-Fc IgG1(reducing); lane 2 was loaded with hu56di-SIRPα(reducing); lane 3 was loaded with AB6.12 IgG1(reducing); lane 4 was loaded with a protein marker; lane 5 was blank; lane 6 was loaded with SIRPα-Fc IgG1(non-reducing); lane 7 was loaded with hu56di-SIRPα(non-reducing); and lane 8 was loaded with AB6.12 IgG1(non-reducing). The results of FIG. 2 demonstrate that the immunoconjugate of the present disclosure was successfully expressed and purified.

FIG. 1a-1b illustrate examples of the immunoconjugate of the present disclosure. Among the above obtained immunoconjugates (e.g., proteinaceous heterodimers), an ate-SIRPα may be an exemplary embodiment of immunoconjugate shown in FIG. 1a , while hu56-Fc6-SIRPα may be an exemplary embodiment of immunoconjugate shown in FIG. 1 b.

Example 4 Binding Kinetics of the Immunoconjugate of the Present Disclosure

Binding kinetics of a hu56di-SIRPα immunoconjugate or control antibodies (SIRPα-Fc IgG1 and AB6.12 IgG1) were determined by the bio-layer inferometry (BLI) method using Octet K2 system (FortéBio). All experiments were performed at 25° C. under a PBS buffer containing 0.5% BSA and 0.5% Tween 20, pH 7.2. CD47-muFc was immobilized onto AHC Biosensors, which were subsequently used in association (with hu56di-SIRPα, SIRPα-Fc IgG1 or AB6.12 IgG1) and dissociation measurements each performed for a time window of 40 seconds and 300 seconds, respectively. After the above steps, the subtracted binding interference data were applied to the calculations of binding constants using the FortéBio analysis software provided with the instrument.

Unexpectedly, hu56di-SIRPα and SIRPα-Fc IgG1 has comparable binding affinity to CD47, as Table 6 depicted.

TABLE 6 Sample KD (M) kon(1/Ms) kdis(1/s) Full X{circumflex over ( )}2 Full R{circumflex over ( )}2 SIRPα-Fc IgG1 5.81E−09 6.50E+05 3.77E−03 0.4161 0.9783 hu56di-SIRPα 5.30E−09 1.14E+06 6.07E−03 0.0924 0.9748 AB6.12 IgG1 1.98E−10 6.51E+05 1.29E−04 0.0349 0.9994

Example 5 FACS Binding Affinity of the Immunoconjugate of the Present Disclosure

The hu56di-SIRPα, SIRPα-Fc IgG1 and AB6.12 IgG1 were compared regarding their binding activities to cell surface human CD47. Briefly, cells expressing CD47-EGFP were incubated with hu56di-SIRPα, SIRPα-Fc IgG1 and AB6.12 IgG1 of various concentrations at 4° C. for 30 min. The cells were then washed to remove any unbound protein and then incubated with an anti-hlgG Fc gamma specific PE antibody (eBiosciences Cat #12-4998-82) at 4° C. for 20 min. The cells were then analyzed by flow cytometry (Life Technology).

As shown in FIG. 3, the horizontal coordinate is the concentration of hu56di-SIRPα, SIRPα-Fc IgG1 and AB6.12 IgG1, and the vertical coordinate is the value of MFI. As expected, the binding affinity of hu56di-SIRPα was much lower than that of SIRPα-Fc IgG1 or AB6.12 IgG1, due to the single arm of SIRPα.

Example 6 In Vitro Pro-Phagocytosis Activity of the Immunoconjugate of the Present Disclosure

Human macrophages were generated by monocytes from Ficoll-purified human peripheral blood mononuclear cells. Monocytes were cultured in media containing human monocyte colony stimulating factor 1 (CSF-1) at 50 ng/ml and human interleukin 10 (IL-10) at 25 ng/ml for at least 1 week to promote development into macrophages. 1×10⁷ Jurket cells were labelled with 5 μM CFSE at 37° C. for 20 min. After labelling, the Jurket cells were incubated with PBS, hu56di-SIRPα or isotype controls (human IgG1) for 15 min at room temperature (RT). 1×10⁵ Jurket cells were then added to the individual wells, mixed with 1×10⁵ macrophage and incubated in a 37° C., 5% CO₂ humidified cell incubator for 2 hours. After the incubation, the wells were washed and the macrophages were analyzed by flow cytometry (Life Technology).

The results are shown in FIG. 4. The horizontal coordinate is PBS, isotype control (human IgG1) and hu56di-SIRPα, and the vertical coordinate is the percentage of phagocytosis. It can be seen that hu56di-SIRPα exhibits strong pro-phagocytosis activity compared with PBS and isotype control group.

Example 7 Dual Antigen Tumor Binding of the Immunoconjugate of the Present Disclosure

Tumor binding affinity of dual antigen and mono antigen were determined by FACS. A375 cell (expressing CD47) and A375-PD-L1 tumor cells (expressing both CD47 and PD-L1) were incubated with various concentration of hu56di-SIRPα at 4° C. for 30 min. Then cells were washed to remove unbound proteins and incubated with an anti-hlgG Fc gamma specific PE antibody (eBiosciences Cat #12-4998-82) at 4° C. for 20 min. The cells were analyzed by flow cytometry (Life Technology).

The results are shown in FIG. 5. The horizontal coordinate is the concentration of hu56di-SIRPα, and the vertical coordinate is the log value of MFI. The result demonstrates that comparing to cells expressing only mono antigen, for cells expressing dual antigens, the binding affinity of hu56di-SIRPα increases significantly.

Example 8 Tumor Binding in Excess Erythrocyte Antigen Sink

An important concern about CD47-based therapeutics is the expression of the target on the surface of erythrocyte, which has the potential to act as a large antigen sink and cause hematological toxicity. Indeed, anemia has been reported in animal treated with SIRPα-Fc IgG1 and CD47-specific antibodies. The binding of hu56di-SIRPα to tumor cell in excess human erythrocytes antigen sink was therefore assessed by flow cytometry. A375 cell (expressing CD47) and A375-PD-L1 tumor cells (expressing both CD47 and PD-L1) were mixed with a 15-fold excess of RBCs, and then incubated with 20 μg/ml hu56di-SIRPα, SIRPα-Fc IgG1 or AB6.12 IgG1 or isotype control (human IgG1) at 4° C. for 30 min. Then the cells were washed to remove unbound proteins and incubated with an anti-hlgG Fc gamma specific PE antibody at 4° C. for 20 min. The cells were analyzed by flow cytometry (Life Technology). The gate of erythrocytes was assessed by anti-human CD235a (eBiosciences Cat #12-9987-80).

The results are shown in FIG. 6 a. 15-folds excess of RBCs are mixed with various concentrations of hu56di-SIRPα, and the binding of RBC is shown. As shown in FIG. 6a , hu56di-SIRPα bound preferentially to tumor cells other than RBC. The MFI of hu56di-SIRPα binding with RBCs was similar to the isotype control. In contrast, SIRPα-Fc IgG1 and AB6.12 IgG1 bound tumor cells and RBCs at the same time. For tumor binding, the results of hu56di-SIRPα were very similar to that of SIRPα-Fc IgG1 and AB6.12 IgG1, when the tumor cells expressed dual antigen.

Similarly, the binding of hu56-SIRPα, ate-SIRPα, hu56-Fc6-SIRPα, SIRPα-Fc IgG1, and AB6.12 IgG1 to tumor cell in excess human erythrocytes antigen sink was therefore assessed by flow cytometry.

A375-PD-L1 tumor cells (expressing both CD47 and PD-L1) were mixed with a 15-fold excess of RBCs, and then incubated with 20 μg/ml hu56di-SIRPα, ate-SIRPα, hu56-Fc6-SIRPα, SIRPα-Fc IgG1, AB6.12 IgG1. or isotype control (human IgG1) at 4° C. for 30 min. Then the cells were washed to remove unbound proteins and incubated with an anti-hlgG Fc gamma specific PE antibody at 4° C. for 20 min. The cells were analyzed by flow cytometry (Life Technology). The gate of erythrocytes was assessed by anti-human CD235a (eBiosciences Cat #12-9987-80).

The results are shown in FIG. 6b . In FIG. 6b , serial number of 1, 2, 3, 4, 5 and 6 correspond to isotype control (human IgG1), hu56di-SIRPα, ate-SIRPα, hu56-Fc6-SIRPα, SIRPα-Fc IgG1 and AB6.12 IgG1, respectively. It was found that hu56di-SIRPα, ate-SIRPα and hu56-Fc6-SIRPα exhibits stronger binding to tumor cells than AB6.12 IgG1. Besides, hu56di-SIRPα, ate-SIRPα and hu56-Fc6-SIRPα almost do not bind to RBC. In comparison, SIRPα-Fc IgG1 bind to RBC and AB6.12 IgG1 exhibiting strong binding to RBC.

Similarly, MCF7 tumor cells were mixed with a 15-fold excess of RBCs, and then incubated with 20 μg/ml Tmab-SIRPα, SIRPα-Fc IgG1, AB6.12 IgG1 or isotype control (human IgG1) at 4° C. for 30 min. Then the cells were washed to remove unbound proteins and incubated with an anti-hlgG Fc gamma specific PE antibody at 4° C. for 20 min. The cells were analyzed by flow cytometry (Life Technology). The gate of erythrocytes was assessed by anti-human CD235a (eBiosciences Cat #12-9987-80).

Similarly, T47D tumor cells were mixed with a 15-fold excess of RBCs, and then incubated with 20 μg/ml 5E5-SIRPα, SIRPα-Fc IgG1, AB6.12 IgG1 or isotype control (human IgG1) at 4° C. for 30 min. Then the cells were washed to remove unbound proteins and incubated with an anti-hlgG Fc gamma specific PE antibody at 4° C. for 20 min. The cells were analyzed by flow cytometry (Life Technology). The gate of erythrocytes was assessed by anti-human CD235a (eBiosciences Cat #12-9987-80).

Similarly, U87 tumor cells were mixed with a 15-fold excess of RBCs, and then incubated with 20 μg/ml mAb806-SIRPα, SIRPα-Fc IgG1, AB6.12 IgG1 or isotype control (human IgG1) at 4° C. for 30 min. Then the cells were washed to remove unbound proteins and incubated with an anti-hlgG Fc gamma specific PE antibody at 4° C. for 20 min. The cells were analyzed by flow cytometry (Life Technology). The gate of erythrocytes was assessed by anti-human CD235a (eBiosciences Cat #12-9987-80).

The results are shown in FIG. 6c-6e . In FIG. 6c , serial number of 1, 2, 3 and 4 correspond to isotype control (human IgG1), SIRPα-Fc IgG1, AB6.12 IgG1, and Tmab-SIRPα, respectively. FIG. 6c shows the binding of isotype control (human IgG1), SIRPα-Fc IgG1, AB6.12 IgG1. and Tmab-SIRPα to MCF7 tumor cells and RBC. In FIG. 6d , serial numbers of 1, 2, 3 and 4 correspond to isotype control (human IgG1), SIRPα-Fc IgG1, AB6.12 IgG1., and 5E5-SIRPα, respectively. FIG. 6 d shows the binding of isotype control (human IgG1), SIRPα-Fc IgG1, AB6.12 IgG1., and 5E5-SIRPα to T47D tumor cells and RBC. In FIG. 6e , serial number of 1, 2, 3 and 4 correspond to isotype control (human IgG1), SIRPα-Fc IgG1, AB6.12 IgG1. and mAb806-SIRPα. FIG. 6e shows the binding of isotype control (human IgG1), SIRPα-Fc IgG1, AB6.12 IgG1. and mAb806-SIRPα. It was found that the three heterodimers of Tmab-SIRPα, 5E5-SIRPα and mAb806-SIRPα almost do not bind to RBC, and their binding to corresponding tumor cells is comparable to SIRPα-Fc IgG1 and AB6.12 IgG1.

Similarly, fresh RBC cell was diluted 2500 times, then it was incubated with 20 μg ml⁻¹ isotype control (human IgG1), hu56di-SIRPα, hu56di-CV1, SIRPα-Fc IgG1 and AB6.12 IgG1, respectively. The results were shown in FIG. 6f , in which serial numbers of 1, 2, 3, 4 and 5 correspond to isotype control, hu56di-SIRPα, hu56di-CV1, SIRPα-Fc IgG1 and AB6.12 IgG1. FIG. 6f shows that hu56di-CV1 exhibits stronger binding to RBC than hu56di-SIRPα, hu56di-CV1 exhibits comparable binding to RBC with AB6.12 IgG1, and SIRPα-Fc IgG1 exhibits weaker binding to RBC than hu56di-CV1 and AB6.12 IgG1. Thus, it can be inferred that hu56di-SIRPα exhibits better safety than hu56di-CV1, SIRPα-Fc IgG1 and AB6.12 IgG1.

Similarly, MC38 tumor cells were mixed with a 15-fold excess of mouse RBCs, and then incubated with 20 μg/ml, a-mPD-L1, mSIRPα-Fc, CV1-Fc, mPD-L1-CV1, mPD-L1-mSIRPα or isotype control (human IgG1) at 4° C. for 30 min. Then the cells were washed to remove unbound proteins and incubated with an anti-hlgG Fc gamma specific PE antibody at 4° C. for 20 min. The cells were analyzed by flow cytometry (Life Technology). The gate of erythrocytes was assessed by anti-mouse RBC antibody (abcam Cat #ab106101). The results are shown in FIG. 6g and FIG. 6h . In FIG. 6g , serial numbers of 1, 2, 3 and 4 correspond to isotype control (human IgG1), mSIRPα-Fc, a-mPD-L1 and mPD-L1-mSIRPα. In FIG. 6h , serial numbers of 1, 2, 3 and 4 correspond to isotype control (human IgG1), CV1-Fc, a-mPD-L1 and mPD-L1-CV1. FIG. 6g and FIG. 6h show that mPD-L1-CV1 exhibits stronger binding to RBC than mPD-L1-mSIRPα, mPD-L1-CV1 exhibits comparable binding to RBC with CV1-Fc, and mSIRPα-Fc exhibits weaker binding to RBC than mPD-L1-CV1. Thus, it can be inferred that mPD-L1-mSIRPα exhibits better safety than mPD-L1-CV1 and mSIRPα-Fc.

Example 9 Platelets Binding of the Immunoconjugate of the Present Disclosure

Platelets also express CD47 as self-antigen to prevent phagocytosis of macrophage. It has been reported that SIRPα-Fc induced thrombocytopenia after being injected into human. Hence, platelets binding of hu56di-SIRPα was examined using flow cytometry. Platelets were separated from fresh blood by centrifugation. Firstly, fresh blood was centrifuged at 200 g for 10 min to get platelet-rich plasma (PRP), then PRP was centrifuged at 1800 g for 10 min to get platelets. The platelets were incubated with 20 μg/ml of hu56di-SIRPα, SIRPα-Fc IgG1 or AB6.12 IgG1 or isotype control (human IgG1) at 4° C. for 30 min. Then, the platelets were washed to remove unbound proteins and incubated with an anti-hlgG Fc APC antibody at 4° C. for 20 min. The platelets were then incubated with anti-human CD42b PE antibody at 4° C. for 20 min. The platelets were analyzed by flow cytometry (Life Technology). The gate of platelets was assessed by anti-human CD42b (Biolegend Cat #303906). The median fluorescence intensity (MFI) of APC in CD42b-positive population indicated drug binding with platelets.

The results are shown in FIG. 7. In FIG. 7a , the horizontal coordinate is the concentration of hu56di-SIRPα, SIRPα-Fc IgG1, AB6.12 IgG1 or the isotype control, and the vertical coordinate shows the MFI value. The quantified results are shown in FIG. 7b . As can be seen from FIG. 7, the binding of hu56di-SIRPα with the platelets was much lower than that of SIRPα-Fc IgG1 and AB6.12 IgG1.

Example 10 In Vivo Anti-Tumor Efficacy and Safety

The CD47 binding site on SIRPα is located in the IgV loop domain. The extracellular IgV domain is relatively well conserved (>75%) in both mouse and human SIRPα, but the binding to CD47 is species specific. Human SIRPα bound strongly to CD47 of NOD mice, but did not bind to CD47 of C57BL/6 mice. Hence, a mPD-L1-mSIRPα was constructed to evaluate anti-tumor efficacy in MC38 syngeneic model.

The results are shown in FIGS. 8a-8d . The horizontal coordinate is the time after tumor inoculation, and the vertical coordinate is the value of tumor volume (FIG. 8a and FIG. 8b ), the percentage of survival (FIG. 8c ), and the fmol of Hemin (FIG. 8d ). C57BL/6 mice were inoculated with MC38 tumor cells. When tumors were well established, human IgG isotype control, a-mPD-L1, mSIRPα-Fc or mPD-L1-mSIRPα was intratumorally administered and tumor growth was monitored. Comparing to the isotype control (hIgG1), the mPD-L1 antibody, and the mSIRPα-Fc, mPD-L1-mSIRPα significantly inhibited tumor growth (FIG. 8a ).

For clinical administration, to evaluate whether systemic treatment with mPD-L1-mSIRPα could inhibit tumor growth, isotype control, mPD-L1-mSIRPα and a mixture of a-mPD-L1 antibody with mSIRPα-Fc was intraperitoneally administered into established tumor-bearing mice and tumor growth was monitored. Comparing to human IgG isotype control and the mixture of mPD-L1 antibody with mSIRPα-Fc, systemic mPD-L1-mSIRPα treatment consistently and significantly slowed tumor growth and prolonged the survival of tumor-bearing mice (FIGS. 8b and 8c ). To determine whether or not mPD-L1-mSIRPα could selectively bind to tumor cells while protecting RBC in vivo, hemin in serum of tumor-bearing mice treated with human IgG isotype control, a-mCD47, mSIRPα-Fc or mPD-L1-mSIRPα was measured. Comparing to a-mCD47 and mSIRPα-Fc treatment, mPD-L1-mSIRPα protected RBC from immune lysis (FIG. 8d ).

Similarly, mPD-L1-mCD47, mPD-L1-mSIRPα and mPD-L1-CV1 were constructed to evaluate anti-tumor efficacy in MC38 syngeneic model.

In FIG. 8e , serial number 1, 2, 3 and 4 correspond to isotype control (hIgG1), mPD-L1-mCD47, mPD-L1-mSIRPα and mPD-L1-CV1. In FIG. 8f , serial number 1, 2, 3, 4 and 5 correspond to untreated sample, isotype control (hIgG1), mPD-L1-mCD47, mPD-L1-mSIRPα and mPD-L1-CV1. FIG. 8e and FIG. 8f show that mPD-L1-mSIRPα and mPD-L1-CV1 exhibit no therapeutic difference in big dose since small but enough mPD-L1-CV1 can reach tumor and bind with tumor cells with higher affinity although mPD-L1-CV1 has a higher binding with RBCs. FIG. 8g shows that mPD-L1-mSIRPα is better than mPD-L1-CV1 in small dose since mPD-L1-CV1 cannot reach tumor due to its higher binding with RBCs.

Similarly, Tmab-mSIRPα, mSIRPα-Fc, IgG1 Isotype and KN034 (Herceptin) were constructed to evaluate anti-tumor efficacy in MC38-HER2 syngeneic model with C57BL/6 mouse. Table 7 provides the specific treatment information for above-mentioned drugs. FIG. 8h shows that Tmab-mSIRPα inhibited tumor growth obviously better than mSIRPα-Fc, IgG1 Isotype and KN034 (Herceptin). FIG. 8i shows the body weight of the mice after tumor implantation.

TABLE 7 Treatment Dose (mg/kg) Dosing Route Schedule IgG1 isotype 6.55 i.p BIW × 3 dose KN034 (Herceptin) 6.55 i.p BIW × 3 dose mSIRPα-Fc 3.55 i.p BIW × 3 dose Tmab-mSIRPα 5 i.p BIW × 3 dose

Example 11 Combination with Chemotherapy

Further, potential synergistic effects of the immunoconjugate of the present disclosure with chemotherapy were tested.

Then, the effects of mPD-L1-mSIRPα in combination with doxorubicin were examined. As shown in FIG. 9, the horizontal coordinate is the day after the treatment, and the vertical coordinate is the percentage of survival of the mice after the treatment. C57BL/6 mice was inoculated with MC38 tumor cells, 11 days later, doxorubicin was administered intratumorally, and mPD-L1-mSIRPα was administered 1 day later. Comparing to the isotype control, doxorubicin alone, or mPD-L1-mSIRPα alone, the combination of doxorubicin with mPD-L1-mSIRPα further significantly inhibited tumor growth and prolonged survival of tumor-bearing mice (FIG. 9).

Example 12 In Vivo Anti-Tumor Efficacy in Humanized Mouse Models

In vivo anti-tumor efficacies of the immunoconjugate of the present disclosure were also tested in humanized mouse models.

Humanized NSG-SGM3 mice were obtained from Jackson Lab, female, 3 wks old. Tumor implant was performed by 12 wks of age. The humanized NSG-SGM3 mice were respectively inoculated with 2×10⁶ MDA-MB-231 tumor cells i.v. on day 0. Then, starting from day 5, 5 doses of the hu56di-SIRPα or isotype control (human IgG1) were administrated to the tumor-bearing humanized NSG-SGM3 mice respectively (for each agent, two doses were administered per week). Then, tumor volume was measured, and the results are shown in FIG. 10. FIG. 10 shows the tumor volume of humanized NSG-SGM3 mice after being administrated with hu56di-SIRPα or isotype control. In FIG. 10, the horizontal ordinate is the days after inoculation, while the vertical ordinate is the tumor volume. These results demonstrate that the immunoconjugate of the present disclosure effectively inhibits tumor growth in vivo.

The effects of the other immunoconjugates (e.g., Tmab-SIRPα, Pmab-SIRPα, Mab806-SIRPα, 5E5-SIRPα, hu56-Fc6-SIRPα, ate-SIRPα, ave-SIRPα and dur-SIRPα) of the present disclosure were also tested, and similar results were observed.

While preferred embodiments of the present application have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the application be limited by the specific examples provided within the specification. While the application has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the application. Furthermore, it shall be understood that all aspects of the application are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the application described herein may be employed in practicing the application. It is therefore contemplated that the application shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the application and that methods and structures within the scope of these claims and their equivalents be covered thereby. 

What is claimed is:
 1. An immunoconjugate comprising: a) a targeting moiety, wherein said targeting moiety exhibits binding specificity to a tumor associated antigen; b) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein said first Fc subunit associates with said second Fc subunit to form a dimer; and c) a single copy of a signal-regulatory protein alpha (SIRPα). 2-3. (canceled)
 4. The immunoconjugate according to claim 1, wherein said SIRPα comprises an amino acid sequence as set forth in SEQ ID NO: 122 or
 123. 5. The immunoconjugate according to claim 1, wherein said targeting moiety comprises an antigen-binding domain of an antibody and said antibody is selected from the group consisting of: anti-EGFRVIII, anti-HER2/neu, anti-PD-L1 and anti-Muc1.
 6. The immunoconjugate according to claim 1, wherein said targeting moiety is an antigen-binding domain of the anti-PD-L1 antibody.
 7. The immunoconjugate according to claim 1, wherein said anti-PD-L1 antibody is selected from the group consisting of Atezolizumab, Avelumab, Durvalumab, KN035 and hu56.
 8. The immunoconjugate according to claim 1, wherein said Fc domain is an IgG Fc domain.
 9. (canceled)
 10. The immunoconjugate according to claim 1, which is a proteinaceous heterodimer comprising a first member and a second member different from said first member, wherein said first member comprises at least one targeting moiety fused to one of the two subunits of the Fc domain, and said second member comprises said single copy of SIRPα fused to the other one of the two subunits of the Fc domain, and said first Fc subunit associates with said second Fc subunit to form said heterodimer.
 11. The immunoconjugate according to claim 10, wherein said targeting moiety is directly or indirectly fused to the amino-terminal amino acid of one of the two subunits of the Fc domain, and said single copy of SIRPα is directly or indirectly fused to the amino-terminal amino acid of the other one of the two subunits of the Fc domain.
 12. The immunoconjugate according to claim 10, wherein the immunoconjugate comprises two of said targeting moiety, which are a first targeting moiety and a second targeting moiety.
 13. The immunoconjugate according to claim 12, wherein said first targeting moiety is directly or indirectly fused to one of said two subunits of the Fc domain, and said second targeting moiety is directly or indirectly fused to the other one of said two subunits of the Fc domain.
 14. The immunoconjugate according to claim 12, which is a proteinaceous heterodimer comprising a first member and a second member different from said first member, wherein: said first member comprises said first targeting moiety fused to one of the two subunits of the Fc domain; said second member comprises said single copy of SIRPα, said second targeting moiety and the other one of the two subunits of the Fc domain, with said single copy of SIRPα fused to one terminal of the Fc subunit and said second targeting moiety fused to the other terminal of the Fc subunit; and said first Fc subunit associates with said second Fc subunit to form said heterodimer.
 15. The immunoconjugate according to claim 12, wherein said first targeting moiety is directly or indirectly fused to the amino-terminal amino acid of one of the two subunits of the Fc domain, said second targeting moiety is directly or indirectly fused to the amino-terminal amino acid of the other one of the two subunits of the Fc domain, and said single copy of SIRPα is directly or indirectly fused to the carboxy-terminal amino acid of the first or the second Fc subunit.
 16. (canceled)
 17. The immunoconjugate according to claim 1, wherein said first Fc subunit comprises a first modification, the second Fc subunit comprises a second modification, and the first modification and the second modification comprise an amino acid substitution at a group of positions selected from any of the following groups: 1) the first modification: Y349 and T366; and the second modification: D356, T366, L368, Y407 and F405; 2) the first modification: Y349, T366 and F405; and the second modification: D356, T366, L368 and Y407: 3) the first modification: Y349, T366 and K409; and the second modification: D356, T366, L368, Y407 and F405; 4) the first modification: Y349, T366, F405, K360 and Q347; and the second modification: D356, T366, L368, Y407 and Q347; 5) the first modification: Y349, T366, F405 and Q347; and the second modification: D356, T366, L368, Y407, K360 and Q347; 6) the first modification: Y349, T366, K409, K360 and Q347; and the second modification: D356, T366, L368, Y407, F405 and Q347; 7) the first modification: Y349, T366, K409 and Q347; and the second modification: D356, T366, L368, Y407, F405, K360 and Q347; 8) the first modification: T366, K409 and K392; and the second modification: T366, L368, Y407, D399 and F405; 9) the first modification: T366 and K409; and the second modification: T366, L368, Y407 and F405; 10) the first modification: T366, K409 and Y349; and the second modification: T366, L368, Y407, F405 and E357; 11) the first modification: T366, K409, Y349 and S354; and the second modification: T366, L368, Y407, F405 and E357; 12) the first modification: T366 and F405; and the second modification: T366, L368, Y407 and K409; 13) the first modification: T366, F405 and D399; and the second modification: T366, L368, Y407, K409 and K392; 14) the first modification: T366, F405 and Y349; and the second modification: T366, L368, Y407, K409 and E357; 15) the first modification: T366, F405, Y349 and S354; and the second modification: T366, L368, Y407, K409 and E357; wherein the position of the amino acid is determined according to the EU index of the KABAT number.
 18. The immunoconjugate according to claim 17, wherein said single copy of SIRPα is directly or indirectly fused to said second Fc subunit comprising said second modification.
 19. An isolated nucleic acid or isolated nucleic acids encoding the immunoconjugate according to claim
 1. 20. A vector or vectors comprising the isolated nucleic acid or isolated nucleic acids according to claim
 19. 21. An isolated host cell comprising the isolated nucleic acid or isolated nucleic acids according to claim
 19. 22. A method for producing an immunoconjugate according to claim 1, comprising (i) culturing a host cell comprising an isolated nucleic acid or isolated nucleic acids encoding the immunoconjugate under conditions to effect expression and formation of the immunoconjugate, and (ii) harvesting the immunoconjugate formed.
 23. A pharmaceutical composition comprising an effective amount of an immunoconjugate according to claim 1, and optionally a pharmaceutically acceptable excipient.
 24. The pharmaceutical composition according to claim 23, further comprising an effective amount of an additional therapeutically active component for cancer treatment, wherein said additional therapeutically active component is a cytotoxic agent for chemotherapy. 25-26. (canceled)
 27. The pharmaceutical composition according to claim 24, wherein said cytotoxic agent is doxorubicin.
 28. (canceled)
 29. A method of treating cancer, comprising administrating the immunoconjugate according to claim 1 in combination with a therapeutically active cytotoxic agent for chemotherapy to a subject in need thereof.
 30. (canceled)
 31. The method according to claim 29, wherein said cancer is selected from the group consisting of: breast cancer, melanoma, and colon cancer.
 32. An isolated host cell comprising the vector or vectors according to claim
 20. 33. A method for producing an immunoconjugate according to claim 1, comprising (i) culturing a host cell comprising a vector or vectors comprising an isolated nucleic acid or isolated nucleic acids encoding the immunoconjugate under conditions to effect expression and formation of the immunoconjugate, and (ii) harvesting the immunoconjugate formed. 